CN118302517A

CN118302517A - Method for producing sheet-like retinal tissue

Info

Publication number: CN118302517A
Application number: CN202280076250.6A
Authority: CN
Inventors: 万代道子; 高桥政代; 山崎优; 堀内茉莉
Original assignee: Sumitomo Pharmaceuticals Co Ltd; RIKEN Institute of Physical and Chemical Research
Current assignee: Sumitomo Pharmaceuticals Co Ltd; RIKEN Institute of Physical and Chemical Research
Priority date: 2021-11-19
Filing date: 2022-11-18
Publication date: 2024-07-05
Also published as: TW202330906A; KR20240103018A; IL312856A; WO2023090427A1; CA3238664A1; AU2022390766A1

Abstract

A method for producing a cell aggregate comprising a retinal tissue having a layered structure, which comprises the step of subjecting a dispersed retinal cell line population comprising 1 or more cells selected from the group consisting of retinal precursor cells and retinoic cell precursor cells to suspension culture or adhesion culture in a medium containing a Wnt signal transduction pathway-acting substance.

Description

Method for producing sheet-like retinal tissue

Technical Field

The present invention relates to a method for producing a sheet-like retinal tissue and a sheet-like retinal tissue.

Background

In recent years, it has been reported that when a properly differentiated stage of the precursor cells of the cells are transplanted into the normal retina of a mouse living body, the precursor cells can survive functionally (non-patent document 1), and the possibility of transplantation treatment for a degenerative disease of the cells such as retinitis pigmentosa has been shown.

There have been many reports on methods for inducing a stereoscopic retinal tissue from the differentiation of pluripotent stem cells obtained by autologous tissue culture, and it has been gradually possible to produce a stereoscopic retinal tissue having a layered structure and to perform transplantation. For example, the following methods are reported: a method of obtaining a multi-layered retinal tissue from pluripotent stem cells (non-patent document 2 and patent document 1); a method in which aggregates of uniform pluripotent stem cells are formed in a serum-free medium containing a Wnt signal transduction pathway-inhibiting substance, the obtained aggregates are subjected to suspension culture in the presence of a basement membrane standard, and then subjected to suspension culture in a serum medium to obtain a multi-layered retinal tissue (non-patent document 3 and patent document 2); and a method of obtaining retinal tissue by suspension-culturing an aggregate of pluripotent stem cells in a medium containing a BMP signaling pathway-acting substance (non-patent document 4 and patent document 3). However, these retinal tissues are produced in the form of spheroid (sphere) -like cell aggregates, and a method for producing sheet-like (flattened) retinal tissues is not known.

On the other hand, wnt2b has been reported to exhibit an effect on the formation of retinal epithelial structures in chickens (non-patent document 5), and the same effect has not been reported in organisms other than chickens.

Prior art literature

Patent literature

Patent document 1: international publication No. 2011/055855

Patent document 2: international publication No. 2013/077425

Patent document 3: international publication No. 2015/025967

Non-patent literature

Non-patent literature 1：Maclaren RE et al.,"Retinal Repair by Transplantation of Photoreceptor Precursors",Nature,444,203-207,(2006)

Non-patent literature 2：Eiraku M.et al.,"Self-organizing optic-cup morphogenesis in three-dimensional culture",Nature,472,51-56,(2011)

Non-patent literature 3：Nakano T.et al.,"Self-formation of Optic Cups and Storable Stratified Neural Retina From Human ESCs"Cell Stem Cell,10(6),771-785,(2012)

Non-patent literature 4：Kuwahara A.et al.,"Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue"Nature Communications,6,6286,(2015)

Non-patent literature 5：Nakagawa,S.et al.,"Identification of the laminar-inducing factor Wnt-signal from the anterior riminduces correct laminar formation of the neural retina in vitro."Developmental Biology,260,414-425,(2003)

Disclosure of Invention

Problems to be solved by the invention

In view of the above, an object of the present invention is to provide a method for reconstructing a neuroepithelial structure of a retinal tissue from retinal cells, a method for producing a sheet-like retinal tissue by applying the method, and a sheet-like retinal tissue. Further, a secondary object is to provide a method for purifying retinal precursor cells, which are more preferable as starting cells, and a method for separating a wide sheet-like retinal tissue produced by using the above method.

Means for solving the problems

The present inventors have conceived to reconstitute a neuroepithelial structure using dispersed retinal cell lines as starting cells, and to produce a sheet-like retinal tissue. However, the following problems are known: by dispersing cells of the retinal system, the polarity of the Apical-Basal (Apical-Basal) cells is destroyed and the neuroepithelial structure cannot be reformed.

As a result of intensive studies, the present inventors have found that the above-described problems can be solved by adding Wnt signaling pathway-acting substances to dispersed retinal cells. In addition, the present inventors have conducted intensive studies in order to reform a more excellent neuroepithelial structure. It was also found that by (1) further adding ROCK inhibitor, SHH signal transduction pathway action substance and/or fibroblast growth factor; (2) In particular, in order to reform a good neuroepithelial structure in a sheet form, culturing is performed on a culture plate coated with an extracellular matrix which becomes a scaffold for cell adhesion; and (3) improving the purity of the retinal precursor cells as starting cells, whereby the foreign cells such as retinal pigment epithelial cells (RPEs) can be removed, thereby completing the present invention.

That is, the present invention relates to the following inventions.

[1] A method for producing retinal tissue having an epithelial structure, which comprises the step of culturing a dispersed population of retinal cells in suspension or in adhesion in a medium containing a Wnt signal transduction pathway-acting substance,

The retinal cell line population contains 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells.

[2] The method according to [1], wherein the Wnt signal transduction pathway-acting substance is 1 or more selected from the group consisting of CHIR99021, BIO, wnt2b and Wnt3 a.

[3] The method according to [1] or [2], wherein the medium further contains 1 or more substances selected from the group consisting of a ROCK inhibitor, an SHH signal transduction pathway-acting substance and an FGF signal transduction pathway-acting substance.

[4] The method according to [3], wherein the ROCK inhibitor is 1 or more selected from the group consisting of Y-27632, fasudil (HA 1077) and H-1152.

[5] The production method according to [3] or [4], wherein the SHH signal transduction pathway-acting substance is 1 or more selected from the group consisting of SAG, PMA and SHH.

[6] The method according to any one of [3] to [5], wherein the substance acting on the FGF signal transduction pathway is 1 or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF 8.

[7] The method according to any one of [1] to [6], wherein the method comprises a step of performing adhesion culture in a medium containing a Wnt signal transduction pathway-acting substance, and the retinal tissue having an epithelial structure is a sheet-like retinal tissue.

[8] The method according to [7], wherein the adhesion culture is performed using a culture vessel coated with an extracellular matrix and/or a temperature-responsive polymer.

[9] The method according to [8], wherein the culture surface of the culture vessel is coated with the temperature-responsive polymer, and the upper surface of the temperature-responsive polymer is coated with the extracellular matrix.

[10] The production method according to [8] or [9], wherein the extracellular matrix is 1 or more selected from the group consisting of collagen, laminin, fibronectin, matrigel (Matrigel) and vitronectin.

[11] The production method according to any one of [8] to [10], further comprising the steps of: the culture vessel coated with the temperature-responsive polymer is exposed to a temperature at which the properties of the temperature-responsive polymer change, whereby the sheet-like retinal tissue is peeled off from the culture vessel.

[12] The production method according to any one of [1] to [11], wherein the production method comprises the steps of: dispersing a cell aggregate containing 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells to obtain the above-described dispersed retinal cell line cell population.

[13] The production method according to [12], wherein the production method comprises the steps of: the pluripotent stem cells are differentiated to obtain the cell aggregate containing 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells.

[14] The production method according to any one of [1] to [13], further comprising the steps of: before suspension culture or adhesion culture is performed on the dispersed retinal cell population, the proportion of retinal precursor cells contained in the dispersed retinal cell population is increased.

[15] The method according to [14], wherein the mixing or differentiation of retinal pigment epithelial cells is inhibited.

[16] The production method according to [14] or [15], wherein the step of increasing the proportion of the retinal precursor cells comprises the steps of: contacting the dispersed population of retinal cells with a substance that binds to more than 1 antigen selected from the group consisting of CD9, CD39, CD90 and CXCR4 to obtain a population of cells expressing the antigen.

[17] The method according to [16], wherein the step of increasing the proportion of the retinal precursor cells comprises the steps of: the dispersed retinal cell population is further contacted with a substance binding to 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84, to obtain a cell population having an expression level of the antigen of the reference value or less.

[18] The method according to any one of [14] to [17], wherein the step of increasing the proportion of the retinal precursor cells comprises:

(1) A step of culturing pluripotent stem cells in the presence of 1 or more selected from the group consisting of Shh signaling pathway-acting substances, ATP and A2A receptor-acting substances to produce cell aggregates;

(2) A step of inducing differentiation of the cell aggregate into a retinal precursor cell;

(3) A step of dispersing the cell aggregate and contacting it with a substance binding to CD 39.

[19] The method according to any one of [1] to [18], wherein the retinal precursor cells and/or the visual cell precursor cells account for 50% or more of the total number of cells contained in the retinal cell population.

[20] The method according to any one of [1] to [18], wherein the retinal precursor cells and/or the visual cell precursor cells account for 80% or more of the total number of cells contained in the retinal cell population.

[21] The method according to any one of [1] to [20], wherein the dispersed retinal cell population is cultured in the medium containing the Wnt signal transduction pathway-acting substance from the start of the suspension culture or the adhesion culture.

[22] The method according to any one of [1] to [21], wherein the orientation of the cells in the epithelial structure is a direction substantially perpendicular to the lamellar direction.

[23] The production method according to any one of [1] to [22], further comprising the steps of: retinal tissue having an epithelial structure obtained by suspension culture or adhesion culture is cut into a size required for transplantation.

[24] The production method according to any one of [1] to [23], wherein the epithelial structure is a multilayer structure.

[25] A sheet-like retinal tissue comprising a retinal cell layer having a multilayer structure, wherein,

(1) The retinal cell-based layer having a multi-layer structure has polarities of basal and apical surfaces,

(2) The above-mentioned retinal cell-based layer having a multilayer structure contains 1 or more cells selected from the group consisting of retinal precursor cells, optic cell precursor cells and optic cells,

(3) In each of the above-mentioned retina cell layers, the orientation of the cells is a direction substantially perpendicular to the layer direction, and

(4) The diameter is more than 8 mm.

[26] The sheet-like retinal tissue according to item [25], wherein the retinal cell-based layer having a multilayer structure further comprises sheet-like retinal pigment epithelial cells bonded to the retinal cell-based layer, the retinal cell-based layer and the surface of each of the sheet-like retinal pigment epithelial cells are substantially parallel in tangential direction, the distal end face of the retinal cell-based layer and the distal end face of the sheet-like retinal pigment epithelial cells face each other, and the retinal cell-based layer and the sheet-like retinal pigment epithelial cells are bonded by an adhesion factor existing therebetween.

[27] The sheet-like retinal tissue according to [26], wherein the adhesion factor is an extracellular matrix or a hydrogel.

[28] The sheet-like retinal tissue according to [27], wherein the adhesion factor is 1 or more selected from the group consisting of gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan and entactin.

[29] The sheet-like retinal tissue according to [27], wherein the adhesion factor is gelatin or fibrin.

[30] A pharmaceutical composition comprising the flaky retinal tissue of any one of [25] to [29 ].

[31] A method of treating a disease based on a disorder of retinal lineage cells or retinal tissue or damage of retinal tissue, comprising the step of transplanting the sheet-like retinal tissue of any one of [25] to [29] to a subject in need of transplantation.

[32] A method of increasing the proportion of retinal precursor cells in a population of cells comprising the step of contacting the population of cells containing retinal precursor cells with a substance that binds to more than 1 antigen selected from the group consisting of CD9, CD24, CD29, CD39, CD47, CD49b, CD49c, CD49f, CD57, CD73, CD82, CD90, CD164, CD200, CD340 and CXCR 4.

[33] The method according to [32], wherein the method comprises a step of contacting the cell population containing the retinal precursor cells with a substance binding to 1 or more antigens selected from the group consisting of CD9, CD39, CD90 and CXCR 4.

[34] The method according to [32] or [33], wherein the method further comprises the step of contacting the cell population containing the retinal precursor cells with a substance binding to 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD 84.

[35] A cell population wherein at least 1 factor selected from the group consisting of CD9, CD39, CD90 and CXCR4 is positive and contains more than 90% of Rx positive retinal precursor cells relative to the total number of cells of the cell population.

[36] The cell population according to [35], wherein 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84 of the retinal precursor cells are negative.

[37] The sheet-like retinal tissue according to any one of [25] to [29], which is used for treating a disease based on a disorder of retinal lineage cells or retinal tissue or damage of retinal tissue.

[38] The use of the sheet-like retinal tissue according to any one of [25] to [29] for producing a therapeutic agent for a disease based on a disorder of retinal cells or retinal tissue or damage of retinal tissue.

Effects of the invention

According to the present invention, a method of reforming a layer structure of a retinal tissue from retinal cells, a method of producing a sheet-like retinal tissue using the method, and a sheet-like retinal tissue can be provided.

Drawings

Fig. 1 is a bright field micrograph showing the reformed state of aggregates from KhES-1 dispersed into single cells and inoculated on day 1 in example 1.

Fig. 2 is a bright field micrograph showing the reformed state of the aggregate from 1231A3 dispersed into single cells and inoculated on day 1 in example 1.

Fig. 3 is a bright field micrograph showing the morphology of aggregates from KhES-1 dispersed into single cells and reformed on day 1 after seeding in example 2.

FIG. 4 is a fluorescence micrograph showing the morphology of aggregates from KhES-1 dispersed into single cells and reformed at day 7 post-inoculation in example 2.

FIG. 5 is a fluorescence micrograph showing the morphology of aggregates from KhES-1 dispersed into single cells and reformed at day 14 post-inoculation in example 2.

FIG. 6 is a graph showing the results obtained by measuring the area of aggregates reformed by single cells derived from KhES-1 in example 2 using Image J. (A) The areas of the aggregates on days 1, 7 and 14 after inoculation are shown, and (B) the ratio (area ratio) of the areas of the aggregates on days 7 and 14 after inoculation to the area of the aggregates on day 1 is shown.

Fig. 7 is a bright field micrograph showing the morphology of aggregates formed on day 1 after dispersing aggregates from 1231A3 into single cells and seeding in example 2.

Fig. 8 is a bright field micrograph showing the morphology of aggregates formed on day 1 after dispersing aggregates from KhES-1 into single cells and seeding in example 3.

FIG. 9 is a fluorescence micrograph showing the morphology of aggregates formed on day 14 after dispersing aggregates from KhES-1 into single cells and seeding in example 3.

Fig. 10 is a bright field micrograph and a fluorescence micrograph showing the morphology of aggregates formed on day 28 after dispersing aggregates from KhES-1 into single cells and seeding in example 3.

FIG. 11 is a photograph showing the results obtained by observing sections of the immunostained (DAPI, rx:: venus, chx 10) aggregates from KhES-1 dispersed into single cells and inoculated with the aggregates on day 15 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 12 is a photograph showing the result of observing an immunostained (. Beta. -catenin) section of an aggregate from KhES-1 dispersed into single cells and inoculated with the aggregate on day 15 after the use of confocal laser scanning fluorescence microscopy in example 3.

FIG. 13 is a photograph showing the result of observation of immunostained (type IV collagen, zo-1) sections of aggregates from KhES-1 dispersed into single cells and inoculated with the aggregates on day 15 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 14 is a photograph showing the results obtained by observing the sections of the immunostained (DAPI, chx10, ki67, pax 6) aggregates obtained by dispersing the aggregates from KhES-1 into single cells and inoculating the single cells on day 28 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 15 is a photograph showing the result of observation of Rx:: venus fluorescence of a section of an aggregate from KhES-1 dispersed into single cells and inoculated on day 28 using a confocal laser scanning fluorescence microscope in example 3.

FIG. 16 is a photograph showing the results of observation of immunostained (type IV collagen, zo-1) sections of aggregates from KhES-1 dispersed into single cells and inoculated with aggregates on day 28 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 17 is a photograph showing the results of observation of sections of the aggregates from KhES-1 dispersed into single cells and immunostained (DAPI, rx:: venus, type IV collagen, zo-1) of the aggregates at day 28 after inoculation using a confocal laser scanning fluorescence microscope in example 3.

FIG. 18 is a photograph showing the results obtained by observing the sections of the immunostained (DAPI, chx10, ki67, pax 6) aggregates obtained by dispersing the aggregates from KhES-1 into single cells and inoculating the single cells on day 28 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 19 is a photograph showing the results of observation of immunostained (CRX, rxRg, NRL, restored protein) sections of aggregates from KhES-1 dispersed into single cells and inoculated with aggregates at day 28 in example 3 using a confocal laser scanning fluorescence microscope.

FIG. 20 is a photograph showing the results obtained by observing the immunostained (DAPI, islet-1, brn3, calomelatin) sections of aggregates from KhES-1 dispersed into single cells and inoculated with aggregates at day 28 in example 3 using a confocal laser scanning fluorescence microscope.

Fig. 21 is a bright field micrograph and a fluorescence micrograph showing the state of aggregates from KhES-1 on days 18, 25, 40, 61 and 75 of differentiation were dispersed into single cells and the aggregates reformed on days 3, 15 and 21 after inoculation in example 4.

FIG. 22 is a photograph of a bright field microscope and a fluorescence microscope (Rx:: venus) showing the state of the aggregates (BMP 4+/BMP 4-) from KhES-1 on day 40 of differentiation in example 5 dispersed into single cells and reformed into aggregates on days 3, 15, and 21 after inoculation.

Fig. 23 is a fluorescence micrograph obtained by inoculating dispersed retinal cells stored in various frozen solutions and observing the morphology of the formed aggregates after 1 day and after 7 days in example 6.

Fig. 24 is a graph showing the viable cell rate (a) after resuscitating dispersed retinal cells stored in various cryopreservation solutions and the area (B) of aggregates reformed using the resuscitated retinal cells in example 6.

Fig. 25 is a photograph showing the result of observation of immunostained sections of a basement membrane expressed in retinal tissue formed by suspension culture using a confocal laser scanning fluorescence microscope in example 7.

Fig. 26 is an immune tissue staining image showing the results obtained by investigating the isoforms of laminin expressed in the neural retinal tissue of a fetal mouse in example 7.

FIG. 27 is a confocal laser scanning fluorescence micrograph showing the confirmation of the reformation of flaky retinal tissue in adhesion culture from a single-cell suspension of retinal lineage cells under conditions 1 to 3 in example 8.

FIG. 28 is a confocal laser scanning fluorescence micrograph showing the confirmation of the reformation of flaky retinal tissue in adhesion culture from a single-cell suspension of retinal lineage cells under conditions 1 to 3 in example 8.

Fig. 29 is a bright field micrograph and a fluorescence micrograph showing the results obtained by confirming the effect of various factors on the reformation of the flaky retinal tissue in the adhesion culture in example 9.

Fig. 30 is a confocal laser scanning fluorescence micrograph showing the results obtained by confirming the effect of various factors in the reformation of the sheet-like retinal tissue in the adhesion culture in example 9.

Fig. 31 is a bright field micrograph, a fluorescence micrograph, and a confocal laser scanning fluorescence micrograph showing the results of the study of the concentration and the addition period of CHIR99021 in the reformation of the flaky retinal tissue in the adhesion culture in example 10.

Fig. 32 is a confocal laser scanning fluorescence micrograph showing the results of the study of the concentration and the addition period of CHIR99021 in the reformation of the sheet-like retinal tissue in the adhesion culture in example 10.

Fig. 33 is a bright field micrograph, bright field and fluorescence micrograph, and fluorescence micrograph showing the results of examining a scaffold inoculated on a Transwell in the reformation of a flaky retinal tissue in an adhesion culture in example 11.

Fig. 34 is a bright field micrograph and a fluorescence micrograph showing the results obtained in example 12 by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in an adhesion culture.

Fig. 35 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 36 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 37 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 38 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 39 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 40 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 41 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 42 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 43 is a confocal laser scanning fluorescence micrograph showing the result obtained by confirming the effect of CHIR99021 in the reformation of a sheet-like retinal tissue in adhesion culture in example 12.

Fig. 44 is a fluorescence micrograph showing the results of examining the effect of maintaining retinal cells by various factors in example 13.

FIG. 45 is a fluorescence micrograph showing the results of a study on conditions effective for maintenance culture of retinal precursor cells in example 14.

Fig. 46 is a stereomicroscope photograph showing the result of re-flaking on collagen in example 15.

FIG. 47 is a fluorescence micrograph showing an implant made from a re-flaked retinal cell sheet in example 16.

Fig. 48 is a fluorescent photomicrograph and a fluorescent photomicrograph showing the observation results of the fundus of the retina after implantation in example 16.

FIG. 49 is a schematic diagram showing the method of example 17 for sorting Rx from a population of cells dispersed into single cells: : graph of FACS analysis results of Venus positive fractions. (A) is a dot pattern, and (B) is a bar pattern.

Fig. 50 is a photograph showing a stereoscopic microscope and a fluorescent microscopic photograph obtained by observing a cell population with/without sorting in example 17.

FIG. 51 is a confocal laser scanning fluorescence micrograph showing a cell population with/without sorting observed in example 17.

FIG. 52 is a confocal laser scanning fluorescence micrograph showing the population of cells with and without sorting observed in example 17.

Fig. 53 is a bright field micrograph and a fluorescence micrograph showing the sorted cell population observed in example 17.

FIG. 54 is a graph showing the Rx observed in example 18 when various proteins were added: : fluorescence microscopy photographs of Venus positive cell populations.

Fig. 55 is a graph showing Rx when various low molecular compounds were added in the observation of example 18: : fluorescence microscopy photographs of Venus positive cell populations.

Fig. 56 is a graph showing Rx observed in example 18 when FGF2, FGF4 and FGF8 are added at different concentrations: : fluorescence microscopy photographs of Venus positive cell populations.

Fig. 57 is a bright field micrograph and a fluorescence micrograph showing the effects of aggregate reformation and retinal differentiation by sorting and addition of FGF8 in example 19.

Fig. 58 is a bright field micrograph and a fluorescence micrograph showing the effects of aggregate reformation and retinal differentiation by sorting and addition of FGF8 in example 19 and a graph showing the results of FACS analysis.

Fig. 59 is a bright field micrograph and a fluorescence micrograph showing the effects of aggregate reformation and retinal differentiation by FGF8 addition in example 19, and a graph showing the results of FACS analysis.

Fig. 60 is a bright field micrograph and a fluorescence micrograph showing the effect of FGF8 addition on re-flaking in example 20.

Fig. 61 is a bright field micrograph and a fluorescence micrograph showing the effect of FGF8 addition on re-flaking in example 20.

Fig. 62 is a bright field micrograph and a fluorescence micrograph showing the effects of re-flaking and retinal differentiation by FGF8 addition in example 20 and a graph showing the results of FACS analysis.

Fig. 63 is a stereoscopic microscope photograph and a fluorescent microscope photograph showing the change with time of the re-flaked retinal sheet in example 21.

FIG. 64 is a diagram showing markers of interest in surface antigen screening in example 22.

FIG. 65 is a graph showing the results of cell sorting using various surface antigens in example 22.

FIG. 66 is a graph showing the results of cell sorting using various surface antigens in example 22.

FIG. 67 is a graph showing the results of cell sorting using various surface antigens in example 22.

FIG. 68 is a graph showing the results of cell sorting using various surface antigens in example 22.

FIG. 69 is a graph showing the results of cell sorting using various surface antigens in example 22.

Fig. 70 is a bright field micrograph and a fluorescence micrograph showing a state of differentiation into brain organoids observed in example 23.

FIG. 71 is a confocal laser scanning fluorescence micrograph showing a state of differentiation into a brain organoid in example 23.

Fig. 72 is a graph showing the results of FACS analysis for expression of CD39 and CD73, CXCR4 in brain organoids in example 23.

Fig. 73 is a bright field micrograph and a fluorescence micrograph showing the results of differentiation induction using SAG at different concentrations in example 24.

Fig. 74 is a graph showing the results of FACS analysis and digitization for CD39 expression and CXCR4 expression after differentiation induction using SAG at different concentrations in example 24.

FIG. 75 is a confocal laser scanning fluorescence micrograph showing an immunostained image of a tissue induced by differentiation using SAG at different concentrations in example 24.

FIG. 76 is a confocal laser scanning fluorescence micrograph showing an immunostained image of a tissue induced by differentiation using SAG at different concentrations in example 24.

Fig. 77 is a confocal laser scanning fluorescence micrograph showing the results of FACS analysis on CD39 expression and BV421 expression after using SAG of different concentrations in example 24 and observation of immunostained images of tissues induced by SAG differentiation of different concentrations.

FIG. 78 is a confocal laser scanning fluorescence micrograph showing an immunostained image of a tissue induced by SAG differentiation at different concentrations in example 24.

FIG. 79 is a confocal laser scanning fluorescence micrograph showing an immunostained image of a tissue induced by SAG differentiation at different concentrations observed in example 24.

FIG. 80 is a graph showing the results of FACS analysis showing the results of the study of substances that can enhance CD39 expression in example 25.

FIG. 81 is a graph in which the results of FACS analysis showing the results of the study of substances that enhance the expression of CD39 in example 25 were digitized. (a) CD39 positive and RX: : results of venus positive, (B) CXCR4 negative and RX: : results of venus positives.

FIG. 82 is a bright field micrograph and a fluorescence micrograph showing the procedure of observing Islet-1 KO hESC-retinal sheets (dd 74) made with FGF 8-and FGF8+ and cutting out grafts for implantation in example 26.

FIG. 83 is a FACS punctate graph comparing with brain organoids and showing the results of screening for various surface antigens for NR in example 27.

FIG. 84 is a FACS punctate showing the results of screening for various surface antigens of NR in example 27.

Fig. 85 is a FACS dot plot showing the results of the temporal change in CD9 expression in hESC-retina in example 28.

FIG. 86 is a FACS dotted graph showing the results of analysis of CD9 and SSEA-1 expression in hESC-retina in example 29.

FIG. 87 is a FACS punctate showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in the hESC-retina in example 30.

Fig. 88 is a FACS dotted diagram showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in example 30.

FIG. 89 is a graph showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in the hESC-retina in example 30.

FIG. 90 is a photograph showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in the hESC-retina in example 30.

Fig. 91 is a photograph showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in example 30.

FIG. 92 is a FACS diagram showing the results of the change over time in the expression of CD9 and SSEA-1 in hESC-retina in example 31.

Fig. 93 is a schematic diagram showing the procedure of a study of RPE sheet and retinal sheet compounding using gelatin in example 32.

Fig. 94 is a stereomicroscope photograph and a fluorescence microscope photograph showing RPE sheets cultured on Transwell and re-flaked neural retinas used in the compounding using gelatin in example 32.

Fig. 95 is a stereoscopic micrograph showing the process of peeling RPE sheets and re-flaked neural retina from Transwell and adding gelatin in example 32.

Fig. 96 is a stereoscopic micrograph showing the procedure of adding gelatin to RPE sheets and re-flaked neural retina in example 32.

Fig. 97 is a stereoscopic and fluorescent micrograph showing the process of complexing RPE sheets with re-flaked neural retina in example 32.

Fig. 98 is a stereoscopic and fluorescent micrograph showing the RPE sheet sandwiched with forceps after complexing with the re-flaked neural retina in example 32.

Fig. 99 is a stereoscopic and fluorescent micrograph showing the RPE sheet composited with the re-flaked neural retina and cut out with scissors in example 32.

Fig. 100 is a stereomicroscope photograph and a fluorescence microscope photograph showing a cross section of the composite RPE sheet and the re-flaked neural retina observed in example 32.

Fig. 101 is a stereoscopic micrograph and a fluorescence micrograph showing the inhalation/ejection of the cut composite RPE sheet and the re-flaked neural retina observed in example 32.

Fig. 102 is a stereoscopic micrograph and a fluorescence micrograph showing RPE sheets cultured on Transwell and re-flaked neural retinas used in the complexing with fibrin in example 33.

Fig. 103 is a stereoscopic and fluorescent micrograph showing the procedure of a compounding study of RPE sheets and re-flaked neural retina detached from Transwell in example 33.

Fig. 104 is a stereoscopic and fluorescent micrograph showing the procedure of the compounding study of recovered RPE sheets and refolding added fibrinogen and thrombin in example 33.

Fig. 105 is a stereoscopic and fluorescent micrograph showing the process of complexing RPE sheets with re-flaked neural retina in example 33.

Fig. 106 is a stereoscopic and fluorescent micrograph showing the process of complexing RPE sheets with re-flaked neural retina in example 33.

Fig. 107 is a stereoscopic and fluorescent micrograph showing the RPE sheet sandwiched with forceps after complexing with the re-flaked neural retina in example 33.

Fig. 108 is a stereoscopic micrograph and a fluorescence micrograph showing a state in which an RPE sheet is composited with a re-flaked neural retina in example 33.

Fig. 109 is a stereomicroscope photograph and a fluorescence microscope photograph showing the process of compounding study in which RPE sheets were peeled from a web of Transwell in example 33.

FIG. 110 is a schematic and photograph showing the results of a study using CELLSHIFTER to remove unwanted hydrogels in example 34.

FIG. 111 is a photograph showing the process of making a planarization sheet and peeling off on a temperature responsive petri dish in example 35.

Detailed Description

[ Definition ]

"Stem cells" refer to undifferentiated cells that have the ability to differentiate and proliferate (particularly, self-replicating). Among stem cells, depending on the differentiation ability, subpopulations including pluripotent stem cells (pluripotent stem cell), multipotent stem cells (multipotent stem cell), multipotent stem cells (unipotent stem cell) and the like are included. Pluripotent stem cells refer to stem cells that are capable of culture in vitro and that have the ability to differentiate into whole cell lineages belonging to the tricodermia (ectodermal, mesodermal, endodermal) and/or extraembryonic tissues (multipotent differentiation (pluripotency)). Multipotent stem cells refer to stem cells that have the ability to differentiate into more than two tissues or cells, but not into all classes of tissues or cells. "pluripotent stem cells" refers to stem cells that have the ability to differentiate into a specific tissue or cell.

The "pluripotent stem cells" may be induced by fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, and the like. Examples of pluripotent stem cells include embryonic stem cells (ES cells: embryonic stem cell), embryonic germ cells (EG cells: embryonic germ cell), and induced pluripotent stem cells (iPS cells: induced pluripotent stem cell). Muse cells (Multi-lineage differentiated sustained stress cells) derived from mesenchymal stem cells (MESENCHYMAL STEM CELL; MSC), GS cells made from germ cells (e.g., testis) are also included in the pluripotent stem cells.

Human embryonic stem cells were established in 1998 and are also beginning to be utilized in regenerative medicine. Embryonic stem cells can be produced by culturing an internal cell aggregate on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described in, for example, WO96/22362, WO02/101057, US5843780, US6200806, US6280718, etc. The embryonic stem cells may be obtained from a predetermined institution or commercially available. For example, human embryonic stem cells KhES-1, khES-2 and KhES-3 are available from Kyoto university of Biotechnology. Human embryonic stem cells Crx: : venus strain and Rx: : the Venus strain (both from KhES-1) is available from national institute of research and development, human physical and chemical research.

The "induced pluripotent stem cell" is a cell obtained by inducing pluripotency by reprogramming (reprogramming) somatic cells by a known method or the like.

Induced pluripotent stem cells were established in 2006 from mountain et al with mouse cells (Cell, 2006, 126 (4), pp.663-676). Induced pluripotent stem cells were also established by human fibroblasts in 2007, and have the same multipotency and self-replication ability as embryonic stem cells (Cell,2007,131(5),pp.861-872;Science,2007,318(5858),pp.1917-1920;Nat.Biotechnol.,2008,26(1),pp.101-106).

Specifically, the induced pluripotent stem cells include cells that are reprogrammed by expressing differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells by any combination of a plurality of genes selected from the reprogramming genome including Oct3/4, sox2, klf4, myc (c-Myc, N-Myc, L-Myc), glis, nanog, sall4, lin28, esrrb, and the like, to induce a multipotency. As a preferred combination of reprogramming factors, (1) Oct3/4, sox2, klf4 and Myc (c-Myc or L-Myc), (2) Oct3/4, sox2, klf4, lin28 and L-Myc (STEM CELLS, 2013; 31:458-466) are exemplified.

As the induced pluripotent stem cells, in addition to a method of manufacturing by direct reprogramming based on gene expression, induced pluripotent stem cells may be induced by somatic cells by addition of a compound or the like (Science, 2013,341, pp.651-654).

In addition, it is also possible to obtain an induced pluripotent stem cell which has been cultured, for example, 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells established by the university of Kyoto the human-induced pluripotent cell line such as 1231A3 cells is available from Kyoto university and iPS A, but is available from Dun Corp. As the induced pluripotent stem cells which have been cultured, for example, ff-I01 cells and Ff-I14 cells and QHJI s04 cells established by Kyoto university can be obtained from Kyoto university.

In the present specification, the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

In the present specification, the pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

Pluripotent stem cells such as human iPS cells can be used for maintenance culture and expansion culture by methods known to those skilled in the art.

"Neural tissue" refers to a tissue composed of cells of a neural system of the brain, midbrain, cerebellum, spinal cord, retina, peripheral nerve, forebrain, hindbrain, telencephalon, metabrain, etc. in the onset or adult stage. The nerve tissue sometimes forms an epithelial structure (nerve epithelium) having a layered structure, and the nerve epithelium in the cell aggregate can be evaluated for the presence amount by bright field observation using an optical microscope.

"Neural cell (Neural cell)" means a cell other than an epidermal system cell in a tissue derived from ectoderm. That is, cells such as a neural precursor cell, a neuron (nerve cell), a glial cell, a neural stem cell, a neuron precursor cell, and a glial cell precursor cell are included. The neural cell also includes cells (retinal cells) constituting the following retinal tissue, retinal precursor cells, retinal layer-specific nerve cells, neural retinal cells, and retinal pigment epithelial cells. The neural cell line can be identified by using neuroepithelial stem cell protein (Nestin), tuJ1, PSA-NCAM, N-cadherin, etc. as markers.

"Neural cells (Neuron Neuronal cell)" are functional cells that form a neural circuit and contribute to information transduction, and can be identified by using the expression of immature neural cell markers such as TuJ1, dcx, huC/D, and/or mature neural cell markers such as Map2, neuN, as an index.

"Neural precursor cells (Neural Precursor cell)" are precursor cell populations comprising neural stem cells, neuronal precursor cells and glial precursor cells, having the ability to proliferate and to produce neurons and glial cells. The neural precursor cells can be identified using, as markers, neuroepithelial stem cell proteins, GLAST, sox2, sox1, musashi, pax6, and the like. Alternatively, cells positive for the markers of the neural cells and positive for proliferation markers (Ki 67, pH3, MCM) can be identified as neural precursor cells.

"Retinal tissue (Retinal tissue)" means a tissue in which one or two or more kinds of retinal cells constituting each retinal layer exist in a certain order in the retina of a living body, and "neural retina (Neural Retina)" means a tissue containing, as a retinal tissue, a neural retinal layer on the inner side other than the retinal pigment epithelium layer among the retinal layers described later.

"Retinal cell line (RETINAL CELL)" refers to the cells or their precursor cells that constitute each retinal layer in the retina of an organism. The retinal lineage cells include, but are not limited to, visual cells (rod cells, cone cells), horizontal cells, amacrine cells, interneuronal cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), mullerian glial cells, retinal Pigment Epithelial (RPE) cells, ciliary bodies, precursor cells of these (e.g., rod precursor cells, bipolar precursor cells, retinal pigment epithelial precursor cells, etc.), neural retinal precursor cells, etc. Among the cells constituting the neural retina layer (also referred to as neural retina cells or neural retina cell (Neural retina-RELATED CELL)), specifically, cells such as visual cells (rod cells, cone cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), mullerian glial cells, and precursor cells of these (for example, rod precursor cells, bipolar precursor cells, etc.) are exemplified. That is, the neural retinal lineage cells do not include retinal pigment epithelial cells and ciliary body cells.

The term "mature retinal lineage cells" refers to cells that can be contained in retinal tissue of a human adult, specifically, differentiated cells such as visual cells (rod cells, cone cells), horizontal cells, amacrine cells, interneuronal cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), mullerian glial cells, retinal Pigment Epithelium (RPE) cells, ciliary cells, and the like. "immature retinal lineage cells" refers to precursor cells (e.g., vision cell precursor cells, bipolar cell precursor cells, retinal precursor cells, etc.) that are identified as differentiating into mature retinal lineage cells.

The "precursor cells of visual cells, precursor cells of horizontal cells, precursor cells of bipolar cells, precursor cells of apocrine cells, precursor cells of retinal ganglion cells, precursor cells of mullerian glial cells, precursor cells of retinal pigment epithelium" refer to precursor cells which are defined to differentiate into visual cells, horizontal cells, bipolar cells, apocrine cells, retinal ganglion cells, mullerian glial cells, retinal pigment epithelium cells, respectively.

"Retinal precursor cells" means: the precursor cells can be differentiated into any immature retinal lineage cells such as a visual cell precursor cell, a horizontal cell precursor cell, a bipolar cell precursor cell, a long process-free cell precursor cell, a retinal ganglion cell precursor cell, a muller glia cell, a retinal pigment epithelial precursor cell, etc., and can be finally differentiated into any mature retinal lineage cells such as a visual cell, a visual rod cell, a cone cell, a horizontal cell, a bipolar cell, a long process-free cell, a retinal ganglion cell, a retinal pigment epithelial cell, etc. "neural retinal precursor cells" means: the precursor cells can be differentiated into any immature neural retinal lineage cells such as a visual cell precursor cell, a horizontal cell precursor cell, a bipolar cell precursor cell, an amacrine cell precursor cell, a retinal ganglion cell precursor cell, a muller glia cell, etc., and can be finally differentiated into any mature neural retinal lineage cells such as a visual cell, a visual rod cell, a visual cone cell, a horizontal cell, a bipolar cell, an amacrine cell, a retinal ganglion cell, etc. Neural retinal precursor cells do not have the ability to differentiate into retinal pigment epithelial cells.

The "retinocyte (photoreceptor cell)" is a layer of retinoic cells present in an organism and has the function of absorbing light stimulus and converting it into an electric signal. The visual cell includes both a cone (cone) that functions in a bright place and a rod (or rod body, rod) that functions in a dark place (referred to as a cone cell, a rod cell, respectively). Examples of the cone cells include cone cells expressing S-opsin and receiving blue light, cone cells expressing L-opsin and receiving red light, and cone cells expressing M-opsin and receiving green light. The visual cell is differentiated and matured from a visual cell precursor cell. Regarding whether or not a cell is an optic cell or an optic cell precursor cell, those skilled in the art can easily confirm, for example, the expression of cell markers (Crx and Blimp1 expressed in an optic cell precursor cell, recovering protein (Recoverin) expressed in an optic cell, rhodopsin expressed in a mature optic cell, S-opsin, M/L-opsin, etc.), the formation of outer segment structures, etc., which will be described later. In one embodiment, the visual cell precursor cells are Crx positive cells and the visual cells are rhodopsin, S-opsin and M/L-opsin positive cells. In one embodiment, the rod cells are NRL and rhodopsin positive cells. In one embodiment, the S-cone cells are S-opsin positive cells, the L-cone cells are L-opsin positive cells, and the M-cone cells are M-opsin positive cells.

The presence of the neural retinal cell line can be confirmed by the presence or absence of expression of a neural retinal cell line-related gene (hereinafter sometimes referred to as "neural retinal cell line marker" or "neural retinal marker"). The presence or absence of expression of the neural retinal cell marker or the proportion of neural retinal cell marker positive cells in the cell population or tissue can be readily confirmed by those skilled in the art. Examples thereof include a method using an antibody, a method using a nucleic acid primer, and a method using a sequencing reaction. As a method of using the antibody, for example, a method such as flow cytometry (FACS) using a commercially available antibody or immunostaining can be used to determine the expression of the protein of the neural retina cell marker by dividing the number of positive cells of the specific neural retina cell marker by the number of total cells. As a method using the nucleic acid primer, for example, RNA expression of a neural retina cell marker can be confirmed by a PCR method, a semi-quantitative PCR method, a quantitative PCR method (e.g., a real-time PCR method). As a method using a sequencing reaction, for example, a nucleic acid sequencer (for example, a next generation sequencer) can be used to confirm RNA expression of a neural retina line cell marker.

Examples of the neural retina cell markers include Rx (also referred to as Rax) and PAX6 expressed in a retinal precursor cell, rx, PAX6 and Chx10 (also referred to as Vsx 2) expressed in a neural retinal precursor cell, crx and Blimp1 expressed in an optic cell precursor cell, and the like. In addition, there may be mentioned Chx10 strongly expressed in bipolar cells, pkcα, go α, VSX1 and L7 expressed in bipolar cells, tuJ1 and Brn3 expressed in retinal ganglion cells, calomelatin and HPC-1 expressed in apocrine cells, calbindin expressed in horizontal cells, restorer proteins expressed in visual cells and precursor cells of visual cells, rhodopsin expressed in visual cells, nrl expressed in visual cells and precursor cells of visual cells, S-opsin and LM-opsin expressed in visual cells, RXR- γ expressed in visual cone cells, precursor cells of visual cone and ganglion cells, trβ2 expressed in visual cone cells or precursor cells thereof appearing in early stages of differentiation in visual cone cells, OTX2 and OC2, pax6 co-expressed in horizontal cells, apocrine cells and ganglion cells, and the like. The surface antigen of the retinal precursor cell or neural retinal precursor cell identified in the examples of the present application may also be used as a marker of the retinal precursor cell or neural retinal precursor cell. Details will be described later.

"Positive cells" refer to cells that express a particular marker on the cell surface or within the cell. For example, "Chx10 positive cells" refer to cells that express the Chx10 protein.

"Retinal pigment epithelial cells" refer to epithelial cells present outside the neural retina in the retina of an organism. Regarding whether or not a cell is a retinal pigment epithelial cell, those skilled in the art can easily confirm, for example, the expression of cell markers (MITF, pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST, TTR, etc.), the presence of melanin particles (blackish brown), tight junctions between cells, polygonal/paving-stone-like characteristic cell morphology, and the like. Whether or not a cell has the function of a retinal pigment epithelial cell can be easily confirmed by the secretion ability of cytokines such as VEGF and PEDF, the phagocytic ability of the extracellular node, and the like. In one embodiment, the retinal pigment epithelial cells are RPE65 positive cells, MITF positive cells, or RPE65 positive and MITF positive cells.

The term "retinal pigment epithelial cell sheet" refers to a single-layer or multi-layer sheet-like structure in which retinal pigment epithelial cells are adhered to each other by biological bonding in at least two dimensions and which is composed of a plurality of cells or two or more cells.

The "retinal layer" refers to each layer constituting the retina, and specifically includes the retinal pigment epithelium layer, the visual cell layer, the outer membrane, the outer particle layer, the outer mesh layer, the inner particle layer, the inner mesh layer, the ganglion cell layer, the nerve fiber layer, and the inner limiting membrane.

The "neural retina layer" refers to each layer constituting the neural retina, and specifically, a visual cell layer, an external membrane, an outer particle layer, an outer mesh layer, an inner particle layer, an inner mesh layer, a ganglion cell layer, a nerve fiber layer, and an inner limiting membrane can be exemplified. The "visual cell layer" refers to a retinal layer formed on the outermost side of the neural retina and containing a large number of 1 or more cells selected from the group consisting of visual cells (rod cells, cone cells), visual cell precursor cells and retinal precursor cells. Layers other than the visual cell layer are referred to as inner layers. The presence or absence of expression of a cell marker, the degree of expression, and the like can be confirmed by a known method, for example, by which retinal layer each cell constitutes.

In the case of retinal tissue in which the proportion of the retinal cells or the retinal precursor cells is small, the layer containing the neural retinal precursor cells to be proliferated is referred to as "neuroblastolayer (neuroblastic layer)", and there are an inner neuroblastolayer (inner neuroblatic layer) and an outer neuroblastolayer (outer neuroblastic layer). The person skilled in the art can determine by a known method, for example, the shade of color under a bright field microscope (outer neuroblastolayer is light and inner neuroblastolayer is dark).

"Ciliary body" includes the "ciliary body", "ciliary body edge area", "Ciliary body" of the process and the body in which it occurs. As markers for "ciliary body" Zic1, MAL, HNF1beta, foxQ1, CLDN2, CLDN1, GPR177, AQP1 and AQP4 are listed. Examples of the "ciliary marginal zone (CILIARYMARGINAL ZONE; CMZ)" include a zone which is a tissue existing in a boundary zone between a neural retina and a retinal pigment epithelium in a retina of a living body and which contains tissue stem cells (retinal stem cells) of the retina. The ciliary body rim area is also known as the ciliary rim (CILIARYMARGIN) or the retinal rim (RETINALMARGIN), and the ciliary body rim area, the ciliary rim and the retinal rim are of equal tissue. The ciliary marginal zone is known to play an important role in supplying retinal precursor cells, differentiating cells, maintaining retinal tissue structure, etc. to retinal tissue. As marker genes for the ciliary marginal zone, for example, rdh gene (positive), otx1 gene (positive) and Zic gene (positive) are cited. By "ciliary body edge area-like structure" is meant a structure similar to the ciliary body edge area.

The "brain tissue" refers to a tissue in which one or more of cells constituting the brain in embryonic or adult stages (for example, brain nerve precursor cells (cortical neural precursor cell), dorsal brain nerve precursor cells, ventral brain nerve precursor cells, brain layer structure-specific nerve cells (neurons), first layer neurons, second layer neurons, third layer neurons, fourth layer neurons, fifth layer neurons, sixth layer neurons, glial cells (astrocytes and oligodendrocytes), precursor cells of these, and the like are arranged in a lamellar three-dimensional manner. The brain in embryonic stage is also called forebrain or telencephalon. The presence of each cell can be confirmed by a known method, for example, the presence or absence of expression of a cell marker, the degree thereof, or the like.

The "brain layer" refers to each layer constituting an adult brain or an embryonic brain, and specifically, molecular layers, exogranular layers, exocone cell layers, endogranular layers, nerve cell layers (endocone cell layers), polymorphous cell layers, first layers, second layers, third layers, fourth layers, fifth layers, sixth layers, cortical regions, intermediate regions, subventricular regions, and ventricular regions (ventricular zones) may be cited.

Examples of the "brain nerve system precursor cells" include neuronal precursor cells, first-layer neuronal precursor cells, second-layer neuronal precursor cells, third-layer neuronal precursor cells, fourth-layer neuronal precursor cells, fifth-layer neuronal precursor cells, sixth-layer neuronal precursor cells, astrocyte precursor cells, oligodendrocyte precursor cells, and the like. Each cell is a precursor cell that is identified as being distinguishable into a first layer of neurons, a second layer of neurons, a third layer of neurons, a fourth layer of neurons, a fifth layer of neurons, a sixth layer of neurons, astrocytes, and oligodendrocytes.

"Brain lineage precursor cells" include multipotent stem cells (multipotent neural stem cells, multi-potent neural stemcell) having the ability to differentiate into at least two or more of the differentiation lineages of a first layer of neurons, a second layer of neurons, a third layer of neurons, a fourth layer of neurons, a fifth layer of neurons, a sixth layer of neurons, astrocytes and oligodendrocytes.

"Brain layer-specific nerve cell" refers to a brain layer-specific nerve cell that is a cell that constitutes the brain layer. Examples of the brain layer-specific neurons include a first layer neuron, a second layer neuron, a third layer neuron, a fourth layer neuron, a fifth layer neuron, a sixth layer neuron, a brain excitatory neuron, and a brain inhibitory neuron.

Examples of brain cell markers include FoxG (alias Bf 1) expressed in brain cells, sox2 and neuroepithelial stem cell proteins expressed in brain nerve line precursor cells, pax6 and Emx2 expressed in dorsal brain nerve line precursor cells, dlx, dlx2 and Nkx2.1 expressed in ventral brain nerve line precursor cells, tbr2, nex, svet1 expressed in neuronal precursor cells, tbr1 expressed in sixth layer neurons, CTIp2 expressed in fifth layer neurons, ROR beta expressed in fourth layer neurons, cux1 or Brn2 expressed in third layer neurons or second layer neurons, and tremelin expressed in first layer neurons (Reelin).

The "cell aggregate" (CELL AGGREGATE) is not particularly limited as long as two or more cells adhere to each other to form a three-dimensional structure, and means, for example, a mass of cells dispersed in a medium such as a medium, a mass of cells formed by cell division, or the like. Cell aggregates also include the situation where a specific tissue is formed.

"Spheroid (sphere) like cell aggregates" refers to cell aggregates having a three-dimensional shape that approximates to a sphere. Examples of the nearly spherical three-dimensional shape include: a spherical shape having a three-dimensional structure and showing, for example, a circle or an ellipse when projected on a two-dimensional plane, and a shape formed by fusing two or more spherical shapes (for example, a shape formed by overlapping 2 to 4 circles or ellipses when projected on a two-dimensional plane). In one embodiment, the aggregate has a vesicular layered structure in the core, and is characterized by dark central portions and bright outer edge portions when observed under a bright field microscope.

The "epithelial tissue" refers to a tissue formed by covering the surface of a body, a lumen (e.g., alimentary canal), a body cavity (e.g., pericardial cavity), etc., without any gap. Cells forming epithelial tissue are referred to as epithelial cells. For epithelial cells, the cells have a apical (apical) -basal (basal) orientation of polarity. The epithelial cells form a firm connection with each other by adhesive (ADHERENCE JUNCTION) and/or tight (tight junction) connections, which can form a cell layer. The tissue formed by overlapping one to more than ten cell layers is epithelial tissue. The tissue from which the epithelial tissue may be formed also includes embryonic and/or adult retinal tissue, brain spinal cord tissue, eye ball tissue, nerve tissue, and the like. The neural retina in this specification is also epithelial tissue. "epithelial structure" refers to a structure characterized by epithelial tissue (e.g., having polarity of basal and apical surfaces).

"Continuous epithelial tissue" refers to tissue having a continuous epithelial structure. The continuous epithelial structure is in a state where the epithelial tissue is continuous. The epithelial tissue is, for example, a state in which 10 cells to 10 ⁷ cells are arranged in a tangential direction with respect to the epithelial tissue, preferably 30 cells to 10 ⁷ cells are arranged in a tangential direction, and more preferably 10 ² cells to 10 ⁷ cells are arranged.

For example, in a continuous epithelial structure formed in retinal tissue, the retinal tissue has a distal end surface unique to the epithelial tissue, and the distal end surface is formed on the surface of the retinal tissue substantially in parallel with at least a visual cell layer (outer particle layer) or the like among the layers forming the neural retinal layer. For example, in the case of a cell aggregate containing a retinal tissue made of pluripotent stem cells, a distal surface is formed on the surface of the aggregate, and visual cells or visual cell precursors of 10 cells or more, preferably 30 cells or more, more preferably 100 cells or more, and even more preferably 400 cells or more are regularly and continuously arranged in a tangential direction to the surface.

In one embodiment, the epithelial tissue is polarized to form a "tip surface" and "basal plane" and "basal membrane". The "basement membrane" refers to a layer (basement membrane) on the basal (basal) side produced by epithelial cells, contains a large amount of laminin and type IV collagen, and has a thickness of 50 to 100 nm. The "substrate surface" refers to a surface (surface layer surface) formed on the "base film" side. The "top surface" refers to a surface (surface layer surface) formed on the opposite side of the "base film". In one embodiment, "top end face" means: in the retinal tissue at the stage of occurrence to the extent that the visual cells or the visual cell precursor cells are observed, an external membrane is formed and the surface is in contact with the visual cell layer (outer particle layer) where the visual cells or the visual cell precursor cells are located. In addition, such a top surface can be identified by immunostaining or the like known to those skilled in the art using an antibody against a marker of the top surface (for example, atypical PKC (hereinafter abbreviated as "aPKC"), a tightly-linked marker (Zo-1), ezrin as ERM protein, E-cadherin, N-cadherin).

[ Method for producing cell aggregate ]

One embodiment of the present invention is a method for producing a cell aggregate containing retinal tissue having an epithelial structure (or a multilayer structure) (in this specification, the term "cell aggregate containing retinal tissue" may be abbreviated as "retinal tissue") from a dispersed retinal cell population. The method comprises the step of culturing the dispersed retinal cell population in suspension or in adhesion in a medium containing a Wnt signal transduction pathway-affecting substance. The dispersed retinal cell population contains 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells. Retinal tissue is as defined above. As one embodiment, the retinal tissue includes a neural retinal tissue, and may include both a neural retinal tissue and a retinal pigment epithelium tissue.

As defined above, in one embodiment, the retinal line cells contain 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells, and may contain cells such as optic cells (rod cells, cone cells), level cells, amacrine cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), and mullerian glia cells. The retinal lineage cells are preferably neural retinal lineage cells. The retinal cell line population used may be derived from a living tissue or from a pluripotent stem cell, and is preferably obtained by differentiation induction of a pluripotent stem cell.

Retinal lineage cells derived from biological tissue can be prepared by those skilled in the art from retinal tissue derived from a living body by well known techniques. Examples include: a method for separating and recovering the eyeballs from RPE after the eyeballs are extracted, and a method for directly extracting the retinas.

One embodiment of the present invention is a method for producing a cell aggregate containing a nerve tissue having an epithelial structure (or a multilayer structure), such as a brain (telencephalon) tissue, from a dispersed nerve cell population. The method comprises the step of carrying out adhesion culture or suspension culture on the dispersed nervous system cell population in a culture medium containing Wnt signal transduction pathway active material. The neural cells preferably contain neural precursor cells (e.g., brain neural precursor cells). The neural cell population used may be derived from a living tissue or from a pluripotent stem cell, and is preferably obtained by differentiation induction of a pluripotent stem cell.

(Method of inducing differentiation of retinal cell line cell population as starting cell)

The pluripotent stem cell-derived retinal lineage cells as starting cells in the production method of the present invention can be obtained by differentiation induction of pluripotent stem cells. As one embodiment, the differentiation-inducing factor may be used to induce differentiation of pluripotent stem cells to obtain aggregates of retinal lineage cells. Examples of the differentiation inducer include a basement membrane standard, a BMP signaling pathway-acting substance, a Wnt signaling pathway-inhibiting substance, and an IGF signaling pathway-acting substance. As one embodiment, a method for producing an aggregate of retinal cells by self-organization is exemplified. Self-organization refers to a mechanism by which a cell population autonomously produces a complex structure. For example, self-organization can be performed by SFEB (Serum-free Floating culture of Embryoid Bodies-LIKE AGGREGATES, serum-free suspension culture of embryoid-like aggregates) methods (WO 2005/12390), SFEBq (WO 2009/148170).

Specific methods of inducing differentiation include WO2011/055855、WO2013/077425、WO2015/025967、WO2016/063985、WO2016/063986、WO2017/183732、PLoS One.2010Jan 20;5(1)：e8763.、Stem Cells.2011Aug;29(8)：1206-18.、Proc Natl Acad Sci USA.2014Jun 10;111(23)：8518-23 and Nat Commun.2014Jun 10;5:4047, but is not particularly limited.

As a specific mode, an aggregate of retinal lineage cells can be prepared by a method including the following steps (a), (B), and (C).

(A) A step of culturing pluripotent stem cells in a medium containing an undifferentiated maintenance factor in the absence of feeder cells;

(B) A step of forming a cell aggregate by subjecting the cells obtained in the step (A) to suspension culture;

(C) A step of further culturing the cell aggregate obtained in the step (B) in suspension in a medium containing a BMP signal transduction pathway active material.

Step (a) may further comprise a tgfβ family signal transduction pathway inhibitor and/or a sonic hedgehog signal transduction pathway inhibitor.

As described later, step (B) may contain a sonic hedgehog signaling pathway action substance and/or a Wnt signaling inhibition substance.

The present method is also disclosed in, for example, WO2015/025967, WO2016/063985, WO2017/183732, and more specifically, reference is made to WO2015/025967, WO2016/063985, WO2017/183732.

The medium used for preparing the aggregates of retinal cells may be a basal medium for cell proliferation (also referred to as basal medium) unless otherwise specified. The basal medium for cell proliferation is not particularly limited as long as it can culture cells, and basal medium sold as a medium for cell proliferation can be used appropriately. Specifically, there may be mentioned, for example, a BME Medium, a BGJb Medium, a CMRL 1066 Medium, a Glasgow MEM (GMEM) Medium, a modified MEM zinc selection Medium, an IMDM Medium, a Medium 199 Medium, a MEM Medium, an Eagle MEM Medium, an αMEM Medium, a DMEM Medium, an F-12 Medium, a DMEM/F12 Medium, an IMDM/F12 Medium, a Ham Medium, an RPMI 1640 Medium, a Fischer's Medium, a Leibovitz's L-15 Medium, or a mixed Medium of these, etc., which can be used for culturing animal cells. In addition, a medium to which an N2 medium as a subsidiary medium is added may be used.

The TGF-beta family signal transduction pathway inhibitor is a substance that inhibits the TGF-beta family signal transduction pathway, that is, the signal transduction pathway transduced by the Smad family, and specifically, TGF-beta signal transduction pathway inhibitors (e.g., SB431542, LY-364947, SB505124, A-83-01, etc.), nodal/activin signal transduction pathway inhibitors (e.g., SB431542, A-83-01, etc.), and BMP signal transduction pathway inhibitors (e.g., LDN193189, doxofmorphine, etc.) may be mentioned. These materials are commercially available.

A substance acting on the signal transduction pathway of sonic hedgehog (hereinafter sometimes referred to as "Shh" or "SHH") is a substance which enhances Shh-mediated signal transduction. Examples of the Shh signal transduction pathway-acting substance include Shh, a partial peptide of Shh, PMA (pummorphomine), SAG (Smoothened Agonist), and the like.

The concentration of the tgfβ family signal transduction pathway inhibitor and the hedgehog signal transduction pathway inhibitor may be such that differentiation into cells of the retinal lineage is induced. For example, SB431542 is generally used at a concentration of 0.1 to 200. Mu.M, preferably 2 to 50. Mu.M. A-83-01 is generally used in a concentration of 0.05 to 50. Mu.M, preferably 0.5 to 5. Mu.M. LDN193189 is generally used at a concentration of 1 to 2000nM, preferably 10 to 300 nM. SAG is generally used at a concentration of 1 to 2000nM, preferably 10 to 700 nM. The PMA is usually used at a concentration of 0.002 to 20. Mu.M, preferably 0.02 to 2. Mu.M.

The undifferentiated maintenance factor is not particularly limited as long as it has an effect of inhibiting differentiation of pluripotent stem cells. As the undifferentiated maintenance factor widely used by those skilled in the art, FGF signal transduction pathway action substances, tgfβ family signal transduction pathway action substances, insulin and the like can be exemplified. Specific examples of the substance acting on the FGF signal transduction pathway include fibroblast growth factors (e.g., bFGF, FGF4, FGF 8). In addition, as tgfβ family signaling pathway acting substances, tgfβ signaling pathway acting substances and Nodal/activin signaling pathway acting substances are exemplified. Examples of tgfβ signal transduction pathway-acting substances include tgfβ1 and tgfβ2. Examples of the substance acting on the Nodal/activin signal transduction pathway include Nodal, activin A and activin B. In the case of culturing human pluripotent stem cells (human ES cells, human iPS cells), the medium in step (a) preferably contains bFGF as an undifferentiated maintenance factor.

The concentration of the undifferentiated maintenance factor in the medium used in the step (a) is a concentration capable of maintaining the undifferentiated state of the cultured pluripotent stem cells, and can be appropriately set by those skilled in the art. For example, in the case where bFGF is used as an undifferentiated maintenance factor in the absence of feeder cells, the concentration thereof is usually about 4 to 500ng/mL, preferably about 10 to 200ng/mL, and more preferably about 30 to 150 ng/mL.

As feeder cells-free medium containing an undifferentiated maintenance factor, which can be used for culturing pluripotent stem cells, various synthetic media have been developed and sold, and for example, essential 8 medium (Life Technologies Co.). The Essential 8 medium is a DMEM/F12 medium containing L-ascorbic acid-2-magnesium phosphate (64 mg/L), sodium selenite (14. Mu.g/L), insulin (19.4 mg/L), naHCO ₃ (543 mg/L), transferrin (10.7 mg/L), bFGF (100 ng/mL) and TGF-beta family signal transduction pathway action substances (TGF-beta 1 (2 ng/mL) or Nodal (100 ng/mL)) (Nature Methods, 8, 424-429 (2011)) as additives. Examples of the commercially available feeder-free medium include S-medium (DS-access), stemPro (Life Technologies), hESF (Proc. Natl. Acad. Sci. USA. 20088 Sep 9;105 (36): 13409-14), mTESR1 (STEMCELL Technologies), mTESR2 (STEMCELL Technologies), teSR-E8 (STEMCELL Technologies) and StemFit (taste element). By using these in the above-mentioned step (A), the present invention can be easily carried out. By using these media, pluripotent stem cells can be cultured under feeder cells-free conditions. As an example, the medium used in step (a) is a serum-free medium to which no BMP signaling pathway acting substance, wnt signaling pathway acting substance, or Wnt signaling pathway inhibiting substance has been added.

In the culture of pluripotent stem cells under feeder cells-free conditions in step (a), a suitable matrix may be used as a scaffold in order to provide a scaffold for pluripotent stem cells instead of feeder cells. Examples of the matrix that can be used as a scaffold include laminin (Nat Biotechnol 28, 611-615, (2010)), a laminin fragment (Nat Commun 3,1236, (2012)), a basement membrane standard (Nat Biotechnol 19,971-974, (2001)), gelatin, collagen, heparan sulfate proteoglycan, nestin, vitronectin (Vitronin), and the like.

The culture time of the pluripotent stem cells in the step (A) is not particularly limited as long as the culture time is within a range in which the effect of improving the quality of the cell aggregate formed in the step (B) is achieved when the culture is performed in the presence of a TGF-beta family signal transduction pathway inhibitor and/or a sonic hedgehog signal transduction pathway inhibitor (for example, 100nM to 700 nM). In one embodiment, the time period is preferably 2 to 96 hours, more preferably 6 to 48 hours, still more preferably 12 to 48 hours, still more preferably 18 to 28 hours (for example, 24 hours).

The medium used in step (B) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding mixing of chemically undefined components, a serum-free medium is preferably used. To avoid the complexity of the preparation, for example, a serum-free medium to which a serum substitute such as commercially available KSR is added in an appropriate amount may be used. As KSR in serum-free media, it is typically added in an amount of about 1% to about 30%, preferably about 2% to about 20%.

In forming the aggregate, first, dispersed cells are prepared by the dispersion operation of the cells obtained in step (a). Examples of the "dispersed cells" obtained by the dispersion operation include a state in which 7 cells (preferably 8 cells or more) or more are single cells and 2 to 50 cells of 3 cells or less (preferably 2 cells or less) are present. The dispersed cells may be in a state in which there is little adhesion (e.g., surface adhesion) of the cells to each other.

The suspension of the dispersed cells is inoculated into an incubator, and the dispersed cells are cultured under a condition of non-adhesion to the incubator, whereby two or more cells are aggregated to form an aggregate. As one embodiment, a certain amount of stem cells dispersed in each well of a multi-well plate (U-shaped bottom, V-shaped bottom) such as a 96-well plate are added, and when they are subjected to stationary culture, the cells are rapidly aggregated, whereby 1 aggregate is formed in each well (SFEBq method). When cells are cultured in suspension using a 96-well plate, the prepared liquid is added to the wells in such a manner that about 1×10 ³ to about 1×10 ⁵ cells (preferably about 3×10 ³ to about 5×10 ⁴ cells, about 4×10 ³ to about 2×10 ⁴ cells) are placed in each of 1 well, and the plate is left to stand to form aggregates.

In one embodiment, the medium used in step (B) comprises sonic hedgehog signaling pathway affecting material.

That is, as a specific mode, an aggregate of retinal lineage cells can be prepared by a method including the following steps (a), (B), and (C):

(A) A step of culturing pluripotent stem cells in a culture medium containing an undifferentiated maintenance factor in the absence of feeder cells and optionally containing a TGF-beta family signal transduction pathway inhibiting substance and/or a sonic hedgehog signal transduction pathway acting substance;

(B) A step of forming a cell aggregate by subjecting the cells obtained in the step (A) to suspension culture in a medium containing a sonic hedgehog signal transduction pathway-acting substance;

As the sonic hedgehog signaling pathway action substance in the step (B), the sonic hedgehog signaling pathway action substance can be used in the concentration (for example, 10nM to 300 nM). The sonic hedgehog signaling pathway-acting substance is preferably contained in the medium from the beginning of suspension culture. ROCK inhibitors (e.g., Y-27632) may be added to the medium. The cultivation time is, for example, 12 hours to 6 days. In one example, the medium used in step (B) is a medium to which 1 or more (preferably all) selected from the group consisting of BMP signaling pathway working substance, wnt signaling pathway working substance, tgfβ family signaling pathway inhibiting substance, and tgfβ family signaling pathway working substance has not been added.

In the step (B), the expression of the cell surface marker CD39 of the retinal precursor cell or the neural retinal precursor cell (Rx positive cell) described later is increased by culturing in a medium containing a substance acting on the sonic hedgehog signal transduction pathway. In one embodiment, the expression of CD39 is increased by adding SAG at a high concentration (100 nM to 1000nM, for example 300 nM) from the beginning of the suspension culture of step (B) (day 0). By using this method, more CD39 positive cells can be purified when the purification step of the retinal precursor cells or the neural retinal precursor cells is performed using CD39, and the production efficiency is improved. The tissues containing CD39 positive cells obtained by adding 300nM SAG were Chx10 positive, lhx2 positive, and NKX2.1 negative, and thus were retinal tissues. In particular, ventral (CoupTF positive) retinal tissue may be induced. The retinal tissue obtained in step (B) without SAG was able to induce dorsal (ALDH 1A1 positive) retinal tissue.

In step (B), the culture is performed in a medium containing 1 or more selected from the group consisting of ATP, ATP analogues (AMP-PNP, etc.) and an A2A receptor agonist (adenosine, CGS21680 (3- [4- [2- [ [ 6-amino-9- [ (2R, 3R,4S, 5S) -5- (ethylcarbamoyl) -3, 4-dihydroxy-oxolan-2-yl ] purin-2-yl ] amino ] ethyl ] phenyl ] propionic acid, etc.), whereby the proportion of CD 39-positive cells in the induced tissue is also increased.

BMP signaling pathway active substances are substances that can enhance BMP-mediated signaling pathways. Examples of the substance acting on the BMP signal transduction pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2 protein, BMP4 protein and BMP7 protein are available, for example, from R & D Systems company, and GDF7 protein is available, for example, from and photoplethysmography.

The medium used in the step (C) may be, for example, a serum-free medium or a serum medium (preferably a serum-free medium) to which a BMP signal transduction pathway active material has been added. Serum-free medium and serum medium can be prepared as described above. In one example, the medium used in step (C) is a medium to which 1 or more (preferably all) selected from the group consisting of Wnt signaling pathway-acting substances, tgfβ family signaling pathway-inhibiting substances, and tgfβ family signaling pathway-acting substances have not been added. In one example, the medium used in step (C) is a medium to which no sonic hedgehog signaling pathway action substance has been added. The medium used in the step (C) is a medium to which a Wnt signaling pathway-acting substance can be added.

The concentration of the BMP signaling pathway activator may be a concentration capable of inducing differentiation into cells of the retinal lineage. For example, in the case of human BMP4 protein, it is added to the medium at a concentration of about 0.01nM to about 1. Mu.M, preferably about 0.1nM to about 100nM, more preferably about 1nM to about 10nM, still more preferably about 1.5nM (55 ng/mL).

The BMP signal transduction pathway-acting substance may be added about 24 hours after the initiation of the suspension culture in step (a), and may be added to the culture medium within several days (for example, within 15 days) after the initiation of the suspension culture. Preferably, the BMP signal transduction pathway-affecting agent is added to the medium between day 1 and day 15, more preferably between day 1 and day 9, and most preferably at day 3 after the suspension culture is started.

Specifically, for example, a part or the whole of the medium may be exchanged with a medium containing BMP4 on days 1 to 9, preferably days 1 to 3 after the initiation of the suspension culture in the step (B), the final concentration of BMP4 may be adjusted to about 1 to 10nM, and the culture may be carried out in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, and more preferably 2 to 5 days. In order to maintain the BMP4 concentration at the same level, the operation of exchanging part or all of the medium for the BMP 4-containing medium may be performed about 1 or 2 times. Alternatively, the BMP4 concentration may be reduced in stages. For example, the concentration of the BMP signaling pathway working substance (BMP 4) may be maintained 2 to 10 days after the start of the suspension culture in step (B), and then the concentration of the BMP signaling pathway working substance (BMP 4) may be reduced stepwise 6 to 20 days after the start of the suspension culture in step (B).

Culture conditions such as the culture temperature and the concentration of CO ₂ in the steps (A) to (C) are appropriately set. The culture temperature is, for example, about 30℃to about 40℃and preferably about 37 ℃. The concentration of CO ₂ is, for example, about 1% to about 10%, preferably about 5%.

By changing the culture time in the above step (C), retinal lineage cells at various differentiation stages can be produced as retinal lineage cells contained in the cell aggregate. That is, retinal lineage cells in cell aggregates containing immature retinal lineage cells (e.g., retinal precursor cells, optic cell precursor cells) and mature retinal lineage cells (e.g., optic cells) in various ratios can be produced. By extending the incubation time of step (C), the proportion of mature retinal lineage cells can be increased.

The above step (B) and/or step (C) may also use the method disclosed in WO 2017/183732. That is, in step (B) and/or step (C), the cell aggregate may be formed by suspension culture in a medium further containing a Wnt signaling pathway-inhibiting substance.

The Wnt signaling pathway inhibitor used in step (B) and/or step (C) is not particularly limited as long as it inhibits Wnt-mediated signaling, and may be any one of a protein, a nucleic acid, a low-molecular compound, and the like. The Wnt-mediated signal is transduced by the Wnt receptor in the form of heterodimers of Frizzled (Fz) with LRP5/6 (low-density lipoprotein receptor-related protein 5/6 ). Examples of Wnt signaling pathway inhibitory substances include substances that directly act on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that inhibit expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant negative Wnt receptors, etc., wnt antagonists, dkk1, cerberus proteins, etc.), substances that inhibit physiological activities resulting from Wnt receptor-mediated signaling [ CKI-7 (N- (2-aminoethyl) -5-chloroisoquinoline-8-sulfonamide), D4476 (4- [4- (2, 3-dihydro-1, 4-benzobiv ]Low molecular weight compounds such as english-6-yl) -5- (2-pyridyl) -1H-imidazol-2-yl ] benzamide), IWR-1-endo (IWR 1 e) (4- [ (3 ar,4s,7r,7 as) -1, 3a,4,7 a-hexahydro-1, 3-dioxo-4, 7-methyl-2H-isoindol-2-yl ] -N-8-quinolyl-benzamide), IWP-2 (N- (6-methyl-2-benzothiazolyl) -2- [ (3, 4,6, 7-tetrahydro-4-oxo-3-phenylthieno [3,2-d ] pyrimidin-2-yl) thio ] acetamide), and the like ], but the present invention is not limited thereto. One or two or more of these Wnt signaling pathway inhibitors may be contained. CKI-7, D4476, IWR-1-endo (IWR 1 e), IWP-2 and the like are known Wnt signal transduction pathway inhibitors, and commercially available products and the like can be suitably obtained. As Wnt signaling pathway inhibitor, IWR1e is preferably used.

The concentration of the Wnt signaling pathway-inhibiting substance in step (B) may be a concentration that can well induce the formation of cell aggregates. For example, in the case of IWR-1-endo, it is added to the medium in a manner to achieve a concentration of about 0.1. Mu.M to about 100. Mu.M, preferably about 0.3. Mu.M to about 30. Mu.M, more preferably about 1. Mu.M to about 10. Mu.M, still more preferably about 3. Mu.M. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirable to use the inhibitor at a concentration that shows Wnt signaling pathway inhibition activity equivalent to the concentration of IWR-1-endo.

In the step (B), the earlier the Wnt signaling pathway inhibitor is added to the medium, the more preferable. The Wnt signaling pathway inhibitor is added to the medium at the beginning of the suspension culture in step (B), usually within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, and still more preferably at the beginning of the suspension culture in step (B). Specifically, for example, a basal medium to which a Wnt signaling pathway inhibitor is added may be added, and a part or all of the medium may be exchanged with the basal medium. The time for which the Wnt signaling pathway-inhibiting substance acts on the cells obtained in step (a) in step (B) is not particularly limited, and it is preferable to act the Wnt signaling pathway-inhibiting substance after the Wnt signaling pathway-inhibiting substance is added to the culture medium at the beginning of the suspension culture in step (B) to the end of step (B) (immediately before the BMP signaling pathway-inhibiting substance is added). Further preferably, as described later, the Wnt signaling pathway inhibitor is continuously exposed to the Wnt signaling pathway inhibitor even after the completion of step (B) (i.e., during step (C)). As one embodiment, as described later, the Wnt signaling pathway inhibitor is allowed to act continuously even after the completion of step (B) (i.e., during step (C)), and the Wnt signaling pathway inhibitor may act until retinal tissue is formed.

In the step (C), any of the Wnt signaling pathway-inhibiting substances described above may be used, and preferably the same type of Wnt signaling pathway-inhibiting substance as used in the step (B) is used in the step (C).

The concentration of the Wnt signaling pathway-inhibiting substance in step (C) may be a concentration capable of inducing retinal precursor cells and retinal tissues. For example, in the case of IWR-1-endo, it is added to the medium in a manner to achieve a concentration of about 0.1. Mu.M to about 100. Mu.M, preferably about 0.3. Mu.M to about 30. Mu.M, more preferably about 1. Mu.M to about 10. Mu.M, still more preferably about 3. Mu.M. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirable to use the inhibitor at a concentration that shows Wnt signaling pathway inhibition activity equivalent to the concentration of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the medium of step (C) is preferably 50 to 150, more preferably 80 to 120, still more preferably 90 to 110, and even more preferably the same as the concentration of the Wnt signaling pathway inhibitor in the medium of step (B), when the concentration of the Wnt signaling pathway inhibitor in the medium of step (B) is 100.

The time period for adding the Wnt signaling pathway-inhibiting substance to the medium is not particularly limited as long as it is within a range capable of forming an aggregate containing retinal cells or retinal tissues, and preferably the earlier the time period is. Preferably, the Wnt signaling pathway-inhibiting substance is added to the culture medium at the beginning of step (C). More preferably, the Wnt signaling pathway inhibitor is added in step (B) and then continuously contained in the medium in step (C) (i.e., from the beginning of step (B)). It is further preferable that the Wnt signal transduction pathway-inhibiting substance is added at the beginning of the suspension culture in step (B), and then continuously contained in the culture medium in step (C). For example, a BMP signaling pathway inhibitor (e.g., BMP 4) may be added to the culture obtained in step (B) (suspension of aggregates in medium containing Wnt signaling pathway inhibitor).

The time for acting the Wnt signaling pathway inhibitor is not particularly limited, but it is preferable that the Wnt signaling pathway inhibitor is added at the beginning of the suspension culture in step (B), and the time is 2 days to 30 days, more preferably 6 days to 20 days, 8 days to 18 days, 10 days to 18 days, or 10 days to 17 days (for example, 10 days) from the beginning of the suspension culture in step (B). In another embodiment, when the Wnt signaling pathway inhibitor is added at the beginning of the suspension culture in step (B), the Wnt signaling pathway inhibitor is preferably allowed to act for 3 to 15 days (e.g., 5 days, 6 days, 7 days), more preferably 6 to 10 days (e.g., 6 days), at the beginning of the suspension culture in step (B).

After culturing the cell aggregate obtained by the above method in a serum-free medium or serum medium containing the Wnt signaling pathway-acting substance and/or the FGF signaling pathway-inhibiting substance for a period of about 2 to 4 days (step (D)), the cell aggregate is cultured in a serum-free medium or serum medium containing no Wnt signaling pathway-acting substance and no FGF signaling pathway-inhibiting substance for about 30 to 200 days (30 to 150 days, 50 to 120 days, 60 to 90 days) (step (E)), whereby a neural retina containing a ciliary rim region-like structure can also be produced.

As an embodiment, a neural retina containing a ciliary marginal zone-like structure can be produced from the cell aggregates obtained in the steps (a) to (C) and 6 to 30 days, 10 to 20 days (10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days) after the initiation of the suspension culture in the step (B) by the steps (D) and (E).

The Wnt signaling pathway-acting substance is not particularly limited as long as it can enhance Wnt-mediated signaling. Specific Wnt signaling pathway-acting substances include, for example, gsk3β inhibitors (e.g., 6-brominated indirubin-3' -oxime (BIO), CHIR99021, and kenparone, for example, in the case of CHIR99021, a range of about 0.1 μm to about 100 μm, and preferably about 1 μm to about 30 μm is included.

The FGF signal transduction pathway inhibitor is not particularly limited as long as it inhibits FGF-mediated signal transduction. Examples of the FGF signal transduction pathway-inhibiting substance include SU-5402, AZD4547, and BGJ 398. For example, in the case of SU-5402, it is added at a concentration of about 0.1. Mu.M to about 100. Mu.M, preferably about 1. Mu.M to about 30. Mu.M, more preferably about 5. Mu.M.

In one example, the medium used in step (D) is a medium to which 1 or more (preferably all) selected from the group consisting of BMP signaling pathway working substance, wnt signaling pathway inhibitory substance, SHH signaling pathway working substance, tgfβ family signaling pathway inhibitory substance, and tgfβ family signaling pathway working substance has not been added.

Some or all of the above step (E) may be performed using the continuous epithelial tissue maintenance medium disclosed in WO 2019/017492. That is, by culturing using a medium for continuous epithelial tissue maintenance, the continuous epithelial structure of the neural retina can be maintained. As an example, a medium in which a B27 supplement (for example, zemoer technologies, 12587010) is mixed with a Neurobasal medium (for example, zemoer technologies, 21103049) can be used as a medium for continuous epithelial tissue maintenance.

In the culture in the step (E), it is preferable to exchange the medium for continuous epithelial tissue maintenance in stages in order to achieve both differentiation and/or maturation of retinal lineage cells (particularly, visual cells) and maintenance of continuous epithelial structures. For example, the first 10 to 30 days are cultured using a basal medium for cell proliferation (for example, a medium in which 10% fetal bovine serum, 1% N2 supplement and 100. Mu.M taurine are added to DMEM/F12 medium), the next 10 to 40 days are cultured using a mixed medium of a basal medium for cell proliferation and a medium for continuous epithelial tissue maintenance (for example, a medium in which 10% fetal bovine serum, 1% N2 supplement and 100. Mu.M taurine are added to DMEM/F12 medium and a medium in which 10% fetal bovine serum, 2% B27 supplement, 2mM glutamine and 100. Mu.M taurine are added to Neurobasal medium) and the next 20 to 140 days are cultured using a medium for continuous epithelial tissue maintenance (for example, a medium in which 10% fetal bovine serum, 2% B27 supplement, 2mM glutamine and 100. Mu.M taurine are added to Neurobasal medium).

In the case where a basal medium for cell proliferation, a medium for continuous epithelial tissue maintenance or any of these mixed media is used in part or all of the above step (E), a thyroid hormone signal transduction pathway-acting substance may be further contained. By culturing in a medium containing a substance acting on a thyroid hormone signal transduction pathway, an aggregate of retinal cells containing a low proportion of bipolar cells, amacrine cells, ganglion cells, or horizontal cells, and an increased proportion of precursor cells of visual cells can be produced.

In the present specification, the substance acting on the thyroid hormone signal transduction pathway is a substance capable of enhancing signal transduction mediated by thyroid hormone, and is not particularly limited as long as it can enhance the thyroid hormone signal transduction pathway. Examples of the substance acting on the thyroid hormone signal transduction pathway include triiodothyronine (hereinafter, abbreviated as T3), thyroxine (hereinafter, abbreviated as T4), and agonists of thyroid hormone receptor (preferably, TR beta receptor).

Examples of thyroid hormone receptor agonists known to those skilled in the art include a pamphlet of International publication No. 97/21993, a pamphlet of International publication No. 2004/066929, a pamphlet of International publication No. 2004/093799, a pamphlet of International publication No. 2000/039077, a pamphlet of International publication No. 2001/098256, a pamphlet of International publication No. 2003/018515, a pamphlet of International publication No. 2003/084915, a pamphlet of International publication No. 2002/094319, a pamphlet of International publication No. 2003/064369, japanese unexamined patent publication No. 2002-053564, japanese unexamined patent publication No. 2002-370978, japanese unexamined patent publication No. 2000-256190, a pamphlet of International publication No. 2007/009913, a pamphlet of International publication No. 2003/094845, a pamphlet of International publication No. 2002/051805, a diphenylmethane derivative, a pyridazine derivative, and an indole derivative.

When T3 is used as a substance acting on the thyroid hormone signal transduction pathway, it is added to the medium so as to be in the range of 0.1 to 1000nM, for example. Examples thereof include those having a thyroid hormone signaling-enhanced activity corresponding to T3 at a concentration of preferably 1 to 500nM, more preferably 10 to 100nM, still more preferably 30 to 90nM, still more preferably 60 nM. When T4 is used as a substance acting on the thyroid hormone signal transduction pathway, it may be added to the medium so as to be in the range of 1nM to 500. Mu.M, for example. Preferably 50nM to 50. Mu.M, more preferably 500nM to 5. Mu.M. When another thyroid hormone receptor agonist is used, the concentration may be one which shows an activity equivalent to the agonist activity shown by T3 or T4 at the above concentration.

The medium used in step (E) may suitably contain L-glutamine, taurine, serum and the like. In one example, the medium used in step (E) is a medium to which 1 or more (preferably all) selected from the group consisting of BMP signaling pathway action substances, FGF signaling pathway inhibitory substances, wnt signaling pathway action substances, wnt signaling pathway inhibitory substances, SHH signaling pathway action substances, tgfβ family signaling pathway inhibitory substances, and tgfβ family signaling pathway action substances have not been added.

As a specific mode, an aggregate of retinal lineage cells can be prepared by a method including the following steps (a) to (E):

(A) A step of culturing in the absence of feeder cells with a medium containing an undifferentiated maintenance factor and optionally containing a TGF-beta family signal transduction pathway inhibiting substance and/or a sonic hedgehog signal transduction pathway acting substance;

(B) A step of forming a cell aggregate by subjecting the cells obtained in the step (a) to suspension culture in a medium which may contain a Wnt signaling pathway-inhibiting substance and/or a sonic hedgehog signaling pathway-acting substance;

(C) A step of further culturing the cell aggregate obtained in the step (B) in suspension in a medium containing a BMP signal transduction pathway active material;

(D) Culturing the cell aggregate obtained in step (C) in a serum-free medium or a serum medium containing a Wnt signaling pathway-acting substance and/or an FGF signaling pathway-inhibiting substance for a period of about 2 days to 4 days; and

(E) Culturing the cell aggregate obtained in step (D) in a serum-free medium or a serum medium which does not contain any Wnt signal transduction pathway-acting substance or FGF signal transduction pathway-inhibiting substance and may contain a thyroid hormone signal transduction pathway-acting substance for about 30 to 200 days.

As a specific embodiment, the aggregates of retinal lineage cells can be prepared by a method comprising the following steps (a ') to (E'):

(A') culturing the pluripotent stem cells in a medium containing an undifferentiated maintenance factor and containing a TGF-beta family signal transduction pathway inhibiting substance and/or a sonic hedgehog signal transduction pathway acting substance in the absence of feeder cells for 12 to 48 hours;

(B ') a step of forming a cell aggregate by culturing the cells obtained in the step (A') in suspension in a medium containing a Wnt signal transduction pathway inhibitor and/or a sonic hedgehog signal transduction pathway inhibitor for 12 hours to 72 days (24 hours to 48 hours);

(C ') a step of further culturing the cell aggregate obtained in the step (B') in suspension in a medium containing a substance acting on the BMP signal transduction pathway for 8 to 15 days (10 to 13 days);

(D ') a step of culturing the cell aggregate obtained in the step (C') in a serum-free medium or a serum medium containing a Wnt signaling pathway-acting substance and/or an FGF signaling pathway-inhibiting substance for 2 days to 4 days; and

(E ') culturing the cell aggregate obtained in step (D') in a serum-free medium or a serum medium which does not contain any Wnt signal transduction pathway-acting substance or FGF signal transduction pathway-inhibiting substance and may contain a thyroid hormone signal transduction pathway-acting substance for about 10 to 200 days.

Here, the step (E') may include the steps of: culturing with a basal medium for cell proliferation for 10 to 30 days, then culturing with a mixed medium of the basal medium for cell proliferation and a medium for continuous epithelial tissue maintenance containing a thyroid hormone signal transduction substance for 10 to 40 days, and further culturing with a medium for continuous epithelial tissue maintenance containing a thyroid hormone signal transduction substance for 20 to 140 days.

In one embodiment, the step (E') comprises a step of culturing in the presence of a substance acting on the thyroid hormone signal transduction pathway for 20 to 60 days (30 to 50 days).

In one embodiment, the culturing time of the steps (B ') to (E') is 70 to 100 days (80 to 90 days).

The time for collecting the retinal tissue containing the retinal cell line as the starting cell is not particularly limited as long as the retinal tissue contains 1 or more cells selected from the group consisting of retinal precursor cells, neural retinal precursor cells and optic cell precursor cells. Preferably, the retinal tissue contains a large amount of retinal precursor cells and/or neural retinal precursor cells. In the above method, for example, a dispersed retinal cell population as a starting cell can be prepared from retinal tissue 10 to 100 days after the start of suspension culture (preferably 9 to 45 days later) as described later.

(Method of inducing differentiation of a cell population of a neural line as a starting cell)

The neural cells derived from pluripotent stem cells as starting cells in the production method of the present invention can be induced by differentiation induction of pluripotent stem cells. As a method of inducing aggregates of pluripotent stem cells into neural cells or neural tissue by suspension culture, various methods have been reported. For example, a method described in WO2005/123902、WO2009/148170、WO2008/035110、WO2011/055855、WO2016/063985、Cell Stem Cell,3,519-32(2008)、Nature,472,51-56(2011)、Cell Stem Cell,10(6),771-775(2012)、Nature Biotechnology,27(3),275-80(2009)、ProcNatl Acad Sci USA,110(50),20284-9(2013) and the like is known, but is not limited thereto. By applying such various methods for inducing neural cells or neural tissue to the aggregate obtained in step (B) and culturing the aggregate obtained in step (B) under appropriate neural differentiation induction conditions, an aggregate containing neural cells or neural tissue can be produced.

An example of a method for producing a neural cell (for example, a brain cell (brain tissue)) as a starting cell in the production method of the present invention is shown below.

A method for producing brain tissue, comprising the steps of (a ") -C"):

(a ") culturing the pluripotent stem cells in a medium containing 1) a tgfβ family signal transduction pathway inhibitor and/or a sonic hedgehog signal transduction pathway inhibitor, and 2) an undifferentiated maintenance factor in the absence of feeder cells;

(B ") a step of culturing the cells obtained in the step (A") in suspension to form an aggregate of cells; and

(C ") subjecting the aggregate obtained in the step (B") to suspension culture in the absence of a differentiation inducer (e.g., a BMP signaling pathway interacting substance) or in the presence of a differentiation inducer (e.g., a TGF-beta family signaling pathway inhibitory substance and/or a Wnt signaling pathway inhibitory substance) to obtain an aggregate containing a neural tissue (brain tissue).

Steps (a ") and (B") can be performed in the same manner as steps (a) and (B) of the above-described method for producing a retinal tissue.

In the step (C') the culture may be performed in a medium to which a differentiation inducer, for example, a substance that activates or inhibits specific signal transduction such as a BMP signal transduction pathway action substance, is not added. By steps (a ") and (B"), the cell aggregates are destined to differentiate into nerves, and thus spontaneously differentiate into nerves (e.g., brain) by culturing in basal medium.

In the step (C "), the tgfβ family signal transduction pathway inhibitor or the Wnt signal transduction pathway inhibitor may be added alone to the medium as the differentiation inducer, but a combination of both is preferable. These differentiation-inducing factors may be added from step (B ").

As the TGF-beta family signal transduction pathway inhibitor used in the steps (B ") and (C"), TGF-beta signal transduction pathway inhibitor, nodal/activin signal transduction pathway inhibitor and BMP signal transduction pathway inhibitor may be mentioned. These factors are the same as the factors described above. The TGF-beta family signal transduction pathway inhibiting substance is preferably SB431542, A-83-01 or LDN193189.

The Wnt signaling pathway inhibitor used in steps (B ") and (C") is not particularly limited as long as it inhibits Wnt-mediated signaling, and may be any one of a protein, a nucleic acid, a low-molecular compound, and the like. The Wnt-mediated signal is transduced by the Wnt receptor in the form of heterodimers of Frizzled (Fz) with LRP5/6 (low-density lipoprotein receptor-related protein 5/6 ). Examples of Wnt signaling pathway inhibitory substances include substances that directly act on Wnt or Wnt receptors (anti-Wnt antibodies, anti-Wnt receptor antibodies, etc.), substances that inhibit expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant negative Wnt receptors, etc., wnt antagonists, dkk1, cerberus proteins, etc.), substances that inhibit physiological activities resulting from Wnt receptor-mediated signaling [ CKI-7 (N- (2-aminoethyl) -5-chloroisoquinoline-8-sulfonamide), D4476 (4- [4- (2, 3-dihydro-1, 4-benzobiv ]Low molecular weight compounds such as english-6-yl) -5- (2-pyridyl) -1H-imidazol-2-yl ] benzamide), IWR-1-endo (IWR 1 e) (4- [ (3 ar,4s,7r,7 as) -1, 3a,4,7 a-hexahydro-1, 3-dioxo-4, 7-methyl-2H-isoindol-2-yl ] -N-8-quinolyl-benzamide), IWP-2 (N- (6-methyl-2-benzothiazolyl) -2- [ (3, 4,6, 7-tetrahydro-4-oxo-3-phenylthieno [3,2-d ] pyrimidin-2-yl) thio ] acetamide), and the like ], but the present invention is not limited thereto. CKI-7, D4476, IWR-1-endo (IWR 1 e), IWP-2 and the like are known Wnt signal transduction pathway inhibitors, and commercially available products and the like can be suitably obtained. As Wnt signaling pathway inhibitor, IWR1e is preferably exemplified.

The concentration of the Wnt signaling pathway inhibitor may be such that it induces differentiation of cells forming aggregates of pluripotent stem cells into brain cells. For example, in the case of IWR-1-endo, it is added to the culture medium in a concentration of about 0.1. Mu.M to about 100. Mu.M, preferably about 0.3. Mu.M to about 30. Mu.M, more preferably about 1. Mu.M to about 10. Mu.M, still more preferably about 3. Mu.M. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirable to use the agent at a concentration that shows Wnt signaling pathway inhibition activity equivalent to the concentration of IWR1 e.

By the culture, differentiation of the cells forming the aggregate obtained in the step (B ") into the brain lineage precursor cells is induced, and an aggregate containing the brain lineage precursor cells can be obtained. The acquisition of the aggregate containing the brain neural precursor cells can be confirmed by, for example, detecting that the aggregate contains cells expressing FoxG1, lhx2, PAX6, emx2, or the like, which are markers of the brain neural precursor cells. As one embodiment of the step (C "), there is a step of culturing the aggregate formed in the step (B") in suspension in a serum-free medium or a serum medium until the cells expressing FoxG gene start to appear. In one embodiment, the culture in step (C') is performed until FoxG1 is expressed by 20% or more (preferably 30% or more, 40% or more, 50% or more, 60% or more) of the cells contained in the aggregate.

The recovery period of the neural tissue containing the neural cells as the starting cells is not particularly limited as long as the neural tissue (brain tissue) contains the neural precursor cells or the brain neural precursor cells, and preferably contains a large amount of these cells. In the above method, for example, a neural tissue (brain tissue) after 10 to 100 days (preferably 15 to 40 days) after the start of suspension culture may be prepared from a dispersed neural cell population as a starting cell as described below.

(Dispersed retinal or neural cell population)

"Dispersion" refers to separation of cells and tissues into small Cell sheets or Cell aggregates (Cell clusters) (2 to 100 cells, preferably 50 to 30 cells, 20 to 10 cells, 5 cells or less, for example, 2 to 5 Cell aggregates) or single cells by dispersion treatment such as enzyme treatment or physical treatment. The dispersed cell population refers to a mass of a certain number of cell sheets or aggregates of cells or single cells. The term "dispersed retinal cell population" refers to a cell population in a dispersed state, and can be obtained by dispersing aggregates of cells such as a living tissue or a cell aggregate. The dispersed retinal cell population is preferably obtained by dispersing aggregates of the retinal cell lines.

In one embodiment, the dispersion method may be a method capable of dispersing cells in a living state, and examples thereof include a mechanical dispersion treatment, a cell dispersion treatment, and a cytoprotective agent addition treatment. These treatments may be performed in combination. Preferably, the cell dispersion treatment is performed, followed by the mechanical dispersion treatment.

As a method of the mechanical dispersion treatment, a blowing treatment or a scraping operation using a doctor blade can be cited.

Examples of the cell dispersion used in the cell dispersion treatment include solutions containing any of enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, dnase, papain, and chelating agents such as ethylenediamine tetraacetic acid. Commercially available cell dispersions, such as TRYPLE SELECT (manufactured by Life Technologies), trypLE Express (manufactured by Life Technologies), and nerve cell dispersion (FujiFilm), can be used.

When dispersing cells, cell death of the cells can be inhibited by treatment with a cytoprotective agent. Examples of the cytoprotective agent used for the cytoprotective agent treatment include FGF signal transduction pathway-acting substances, heparin, IGF signal transduction pathway-acting substances, and serum or serum substitutes. In addition, in order to inhibit cell death (particularly cell death of human pluripotent stem cells) induced by dispersion, an inhibitor of Rho-related coiled-coil kinase (Rho-associated coiled-coil Containing Protein Kinase ("ROCK" or "Rho kinase")) or an inhibitor of myosin may be added at the time of dispersion. Examples of the ROCK inhibitor include Y-27632, fasudil (HA 1077) and H-1152. As the myosin-inhibiting substance, bupivastatin is exemplified. As a preferred cytoprotective agent, ROCK inhibitors can be mentioned.

The dispersed retinal cell line population (or neural cell line population) may be exemplified by the following states: the cell mass contains single cells (SINGLE CELL), for example, at least 70% and preferably at least 80% of the total number of cells in the cell population are single cells, and at most 30% and preferably at most 20% of the total number of cells in the cell population are 2 to 50 cells. In the dispersed retinal cell line population (or neural cell line population), the adhesion (e.g., facial adhesion) of cells to each other is substantially lost. Further, the dispersed retinal cell line population (or neural cell line population) is more preferably composed of single cells as much as possible, and such a dispersed retinal cell line population (or neural cell line population) can be obtained by removing a cell mass which does not become single cells after the dispersion treatment. The method for removing the cell mass is not particularly limited, and examples thereof include removal of the mass by a filter (cell strainer). In one embodiment, the dispersed cells may be in a state in which cell-cell junctions (e.g., adhesive junctions) are substantially lost.

When a retinal cell population is prepared from retinal tissue as a starting cell, the retinal tissue may contain unwanted cells such as retinal pigment epithelial cells. For example, the case where retinal tissue is collected from a living body, the case where step (D) and step (E) in the method of producing retinal tissue from the pluripotent stem cells are mentioned, and the like. In this case, the area where unwanted cells such as retinal pigment epithelial cells are located can be excised, and the retinal cell population can be dispersed. In the case of retinal pigment epithelial cells, the cells can be discriminated by morphology and pigment, and the excision can be easily performed by a person skilled in the art.

Before the start of the culture in a medium containing a Wnt signaling pathway-acting substance, a step (purification step) for improving the purity of the retinal precursor cells or neural retinal precursor cells may be performed as described below, and further maintenance/expansion culture of the dispersed retinal cell population (or neural cell population) may be performed. The medium is not particularly limited as long as it is a medium (DMEM medium or the like) in which retinal cells (or neural cells) can survive and proliferate. Since the frozen and fused retinal cell population can be used as the starting cells in the production method of the present invention, the dispersed retinal cell population can be frozen and stored. The frozen stock solution is not particularly limited, and commercially available frozen stock solutions can be used.

[ Purification of retinal precursor cells ]

Before the culture of the above-described dispersed retinal cell line population in a medium containing a Wnt signaling pathway-acting substance is started, the dispersed retinal cell line population may be subjected to a step (purification step) for increasing the proportion (purity) of retinal precursor cells, preferably the proportion (purity) of neural retinal precursor cells. In the purification step, operations such as cell sorting may be performed using specific markers expressed in the retinal precursor cells and/or the neural retinal precursor cells. Cell sorting can be performed using known techniques such as FACS and MACS. By performing the purification step of the retinal precursor cells and/or the neural retinal precursor cells, the incorporation of RPE cells and target outer cells in the production method described in the present specification can be reduced. By increasing the proportion of retinal precursor cells contained in the dispersed retinal cell population, the production of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells can be suppressed. Whether or not production of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells is suppressed can be determined based on the markers, morphology, properties, and the like of the retinal pigment epithelial cells after culturing the dispersed retinal cell population by a culture method described later. Inhibiting the production of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells may refer to: the ratio of retinal pigment epithelial cells to the total number of cells after the culture was suppressed as compared with the case where the step of increasing the ratio of retinal precursor cells was not performed. If the ratio of retinal pigment epithelial cells is about the level described in paragraph 0175, it can be determined that the production of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells is inhibited.

As cell markers (positive markers) of retinal precursor cells, rx, chx10 and the like are well known. However, these genes are expressed in cells, and thus, for example, it is necessary to use cells in which the genes are linked to a fluorescent protein by gene recombination techniques, cells in which the genes are replaced with a fluorescent protein, or the like (for example, rx:: venus cells). Thus, positive markers for retinal and/or neural retinal precursor cells were explored and CD9(Genbank ID：NM＿001769.4、NM＿001330312.2)、CD24(Genbank ID：NM＿001291737.1、NM_001291738.1、NM_001291739.1、NM_001359084.1、NM＿013230.3)、CD29(Genbank ID：NM＿002211.4、NM＿033668.2、NM＿133376.2)、CD39(Genbank ID：NM＿001098175.2、NM＿001164178.1、NM_001164179.2、NM_001164181.1、NM_001164182.2、NM_001164183.2、NM_001312654.1、NM_001320916.1、NM_001776.6)、CD47(Genbank ID：NM＿001777.3、NM＿198793.2)、CD49b(Genbank ID：NM＿002203.4)、CD49c(Genbank ID：NM＿002204.4)、CD49f(Genbank ID：NM＿000210.4、NM＿001079818.3、NM＿001316306.2、NM＿001365529.2、NM＿001365530.2)、CD57(Genbank ID：NM＿001367973.1、NM＿018644.3、NM＿054025.3)、CD73(Genbank ID：NM＿001204813.1、NM＿002526.4)、CD82(Genbank ID：NM＿001024844.2、NM＿002231.4)、CD90(Genbank ID：NM＿001311160.2、NM＿001311162.2、NM＿001372050.1、NM＿006288.5)、CD164(Genbank ID：NM＿001142401.2、NM＿001142402.2、NM＿001142403.3、NM＿001142404.2、NM＿001346500.2、NM＿006016.6)、CD200(Genbank ID：NM＿001004196.3、NM＿001318826.1、NM＿001318828.1、NM_001318830.1、NM_001365851.2、NM_001365852.1、NM_001365853.1、NM_001365854.1、NM_001365855.1、NM_005944.7)、CD340(Genbank ID：NM＿001005862.2、NM＿001289936.1、NM＿001289937.1、NM＿001289938.1、NM＿004448.3) and CXCR4 (Genbank ID: NM_ 001008540.2, NM_ 001348056.2, NM_ 001348059.2, NM_ 001348060.2, NM_ 003467.3) were found to be expressed on the cell surface of these cells. As preferred cell surface markers, there may be mentioned, for example, CD9, CD39, CD90 and CXCR4. As one embodiment, a method for increasing the proportion of retinal precursor cells in a cell population may be performed before starting culturing in the medium containing a Wnt signaling pathway-affecting substance, the method comprising the steps of: the above cell population containing retinal precursor cells is contacted with a substance (e.g., antibody, peptide, etc.) that binds to 1 or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4 and positive fractions are separated. By this method, the mixing of RPE cells in the production method described in the present specification can be reduced.

As negative markers of these cells, i.e., markers not expressed in these cells, SSEA1 (Genbank ID: NM_ 002033.3), CD66b (GenbankID: NM_ 001816.4), CD69 (Genbank ID: NM_ 001781.2), and CD84 (Genbank ID: NM_ 001184879.2, NM_ 001184881.2, NM_ 001184882.1, NM_ 001330742.2, NM_ 003874.4) were found. By combining with the above positive markers, the purity of the retinal precursor cells and/or neural retinal precursor cells can be further improved. As one embodiment, a method for increasing the proportion of retinal precursor cells in a cell population may be performed before starting culture in a medium containing a Wnt signaling pathway-affecting substance, the method comprising the steps of: the above-mentioned cell population containing retinal precursor cells is contacted with a substance (e.g., an antibody) that binds to 1 or more antigens selected from the group consisting of CD9, CD39, CD90 and CXCR4, preferably also with a substance (e.g., an antibody) that binds to 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84, and the positive fraction of the marker of the former and the negative fraction of the marker of the latter are separated. The step of contacting the dispersed retinal cell population with a substance (for example, an antibody) that binds to 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84, and separating the negative fractions of these markers is a step of obtaining a cell population in which the expression level of the antigen is equal to or less than a reference value. Here, the reference value may be arbitrarily set by one skilled in the art. For example, a negative fraction can be isolated by obtaining a cell population treated with a fluorescent-labeled antibody against the antigen of the above-mentioned fluorescent-labeled antibody of the same intensity as that of the cell population treated with a fluorescent-labeled isotype control antibody.

The method of increasing the ratio of the retinal precursor cells and/or neural retinal precursor cells in the cell population using a substance (for example, an antibody) that binds to the positive marker and the negative marker is not limited to the method that is one step of the production method described in the present specification. The method of increasing the proportion of retinal precursor cells and/or neural retinal precursor cells using cell surface markers may also be used, for example, as a step in a method of producing other retinal tissues.

In one embodiment, the retinal precursor cells and/or neural retinal precursor cells in the dispersed retinal cell population may be, for example, 30% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more of the total number of cells. Further, for example, the dispersed retinal cell population may contain 50% or more, preferably 80% or more, 85% or more, 90% or more, 95% or more of cells (retinal precursor cells or neural retinal precursor cells) positive for Rx and/or Chx10, which are positive for at least 1 marker selected from the group consisting of CD9, CD39, CD90 and CXCR4, relative to the total number of cells in the cell population. Furthermore, the cell population is preferably negative for 1 or more, preferably 2 or more, 3 or more or all antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD 84. The negative value referred to herein may be equal to or less than the reference value.

In one embodiment, the step of increasing the proportion of retinal precursor cells (purification step) comprises the steps of:

(3) A step of dispersing the cell aggregate and contacting it with a substance (e.g., an antibody) that binds to CD 39.

Details of steps (1) and (2) are described in "method for inducing differentiation of retinal cell lines as starting cells".

(Reformatting the structure by culturing the dispersed population of retinal or neural cells)

The method for producing retinal tissue according to the present invention comprises a step of subjecting a dispersed population of retinal cells to an adherent culture or a suspension culture in a medium containing a Wnt signal transduction pathway-affecting substance. The epithelial structure (multilayer structure) can be reformed from the dispersed retinal cell population by any of the culture methods. In order to produce a sheet-like retinal tissue having an epithelial structure (or a multilayer structure), an adhesion culture is preferable. The production method of the present invention may be similarly carried out using the dispersed neural cell population as the starting cell. The following describes the case of using the dispersed retinal cell population as the starting cell, but the same applies to the case of using the dispersed neural cell population as the starting cell unless otherwise specified.

The medium used for culturing the dispersed retinal cell population is not particularly limited as long as it is a medium in which retinal cells can survive and proliferate. In one embodiment, the medium used for culturing the dispersed retinal cell population includes a medium for maintaining continuous epithelial tissue. As an example, a medium in which a B27 supplement (for example, zemoer technology, 12587010) is mixed with a Neurobasal medium (for example, zemoer technology, 21103049) can be cited as a medium for continuous epithelial tissue maintenance.

The Wnt signaling pathway-acting substance contained in the medium is not particularly limited as long as it is a substance that can enhance Wnt-mediated signaling. Specific examples of Wnt signaling pathway-acting substances include gsk3β inhibitors (for example, 6-brominated indirubin-3' -oxime (BIO), CHIR99021, kenparoone, wnt2b, wnt3a, etc. as Wnt proteins, and partial peptides thereof, etc. may be 1 substance or 2 or more substances.

The concentration of the Wnt signaling pathway-acting substance may be a concentration capable of inducing the formation of a desired cell aggregate (e.g., the reformation of an epithelial structure (or a multilayer structure)). The concentration of the Wnt signaling pathway-acting substance may be, for example, 0.01 μm to 100 μm, preferably 0.1 μm to 10 μm, and more preferably 1 μm to 10 μm when CHIR99021 is used. When another Wnt signaling pathway-acting substance is used, the concentration may be one that shows Wnt signaling activation to the same extent as CHIR99021 at the above concentration. The Wnt signaling can be determined by, for example, confirming the expression of β -catenin.

The timing of adding the Wnt signaling pathway-acting substance is not particularly limited, and it is preferably added as early as possible after initiation of suspension culture or adhesion culture for re-flaking. In one embodiment, it is preferable to culture in a medium containing a Wnt signaling pathway-acting substance from the start of the culture. The number of days of culture in the medium containing the Wnt signaling pathway-acting substance is not particularly limited as long as the effect of reforming the epithelial structure (or multilayer structure) having the polarity of the apical surface/basal membrane can be observed, and is, for example, 1 to 14 days.

In one embodiment, the medium may further contain 1 or more substances selected from the group consisting of ROCK inhibitors, SHH (sonic hedgehog) signal transduction pathway-acting substances, and Fibroblast Growth Factor (FGF) signal transduction pathway-acting substances. For example, the effect of promoting reaggregation of the dispersed retinal precursor cell population can be observed by adding a ROCK inhibitor, the effect of promoting proliferation of cell aggregates and enlarging cell aggregates can be observed by adding an SHH signal transduction pathway-acting substance, and the effect of reducing cell damage and inhibiting differentiation induction into RPE cells can be observed by adding an FGF signal transduction pathway-acting substance (for example, FGF2 and FGF 8).

The ROCK inhibitor is not particularly limited as long as it can inhibit the function of Rho kinase (ROCK), and examples thereof include Y-27632 (for example, refer to Ishizaki et al, mol. Pharmacol.57,976-983 (2000)), narumiya et al, methods enzymol.325,273-284 (2000)), fasudil/HA 1077 (for example, refer to Uenata et al, nature 389:990-994 (1997)), H-1152 (for example, refer to Sasaki et al, pharmacol. Ther.93:225-232 (2002)), wf-536 (for example, refer to Nakajima et al, cancer chemothermo Pharmacol.52 (4): 319-324 (2003)), and derivatives thereof, and antisense nucleic acids, RNA interference-inducing nucleic acids (for example, siRNA), dominant negative mutants, and expression vectors thereof for ROCK. In addition, other low molecular weight compounds are also known as ROCK inhibitors, and thus such compounds or their derivatives may also be used in the present invention (for example, refer to U.S. patent application publication No. 20050209261, U.S. patent application publication No. 20050192304, U.S. patent application publication No. 20040014755, U.S. patent application publication No. 20040002508, U.S. patent application publication No. 20040002507, U.S. patent application publication No. 20030125344, U.S. patent application publication No. 20030087919 and international publication No. 2003/062227, international publication No. 2003/059913, international publication No. 2003/062225, international publication No. 2002/076976, and international publication No. 2004/039796). The ROCK inhibitor may be 1 or 2 or more ROCK inhibitors. The ROCK inhibitor preferably contains 1 or more substances selected from the group consisting of Y-27632, fasudil (HA 1077) and H-1152.

The concentration of the ROCK inhibitor can be appropriately set by those skilled in the art according to experimental conditions. As one embodiment, the concentration may be a concentration capable of promoting reaggregation of the dispersed retinal precursor cell population. For example, when Y-27632 is used, it may be 0.1. Mu.M to 1mM, preferably 1. Mu.M to 100. Mu.M, more preferably 5. Mu.M to 20. Mu.M. When a ROCK inhibitor other than Y-27632 is used, the concentration may be one which exhibits a ROCK inhibiting effect equivalent to that of Y-27632 at the above concentration. The ROCK inhibition can be determined by a person skilled in the art, for example, by expression analysis of MLC2 phosphorylation.

SHH (sonic hedgehog) signaling pathway action substance refers to a substance that can enhance signal transduction mediated by SHH (sometimes also referred to as Shh). Examples of the SHH signal transduction pathway-acting substance include proteins belonging to the hedgehog family (e.g., shh, ihh), SHH receptors, shh receptor agonists, PMA (Purmorphamine; 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl ] -2- (1-naphthoxy) -9H-purin-6-amine), SAG (Smoothened Agonist; N-methyl-N '- (3-pyridylbenzyl) -N' - (3-chlorobenzo [ b ] thiophene-2-carbonyl) -1, 4-diaminocyclohexane), and the like. One or two or more of these substances may be contained as substances acting on the Shh signaling pathway. The Shh signaling pathway-acting substance is preferably 1 or more selected from the group consisting of Shh (Genbank accession number: NM-000193, NP-000184), SAG, and PMA.

The concentration of the SHH signal transduction pathway-acting substance may be appropriately set by those skilled in the art according to experimental conditions. As one embodiment, the concentration is a concentration within a range in which an effect of increasing the cell aggregate can be observed. For example, SAGs are typically used at concentrations of 1 to 2000nM, preferably 10 to 700 nM. The PMA is usually used at a concentration of 0.002 to 20. Mu.M, preferably 0.02 to 2. Mu.M. SHH is generally used at 4 to 500ng/mL, preferably 10 to 200 ng/mL. When another substance acting on the SHH signal transduction pathway is used, the concentration may be one which shows the same level of SHH signal activation as SAG at the above concentration. The man skilled in the art can determine the SHH signal activation by means of, for example, expression analysis of downstream signals (SMO, GLI).

The FGF signaling pathway-acting substance is not particularly limited as long as it is a substance that can enhance FGF-mediated signaling. As the substance acting on the FGF signal transduction pathway, specifically, fibroblast growth factors (e.g., bFGF, FGF4, FGF8, and FGF 9) can be cited. The FGF signal transduction pathway-acting substance is preferably 1 or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF 8.

The concentration of the FGF signaling pathway-acting substance can be appropriately set by those skilled in the art according to experimental conditions. As one embodiment, the concentration is within a range in which an effect of suppressing differentiation induction into RPE cells can be observed. For example, when FGF2, 4 or 8 is used, it is about 4 to 500ng/mL, preferably about 10 to 200ng/mL, and more preferably about 25 to 100 ng/mL. When other FGF signal transduction pathway-acting substances are used, the concentration may be a concentration that exhibits FGF signal activation to the same extent as that of FGF8 or the like at the above concentration. FGF signaling can be determined by one skilled in the art, for example, by expression analysis of downstream signals (Akt, MEK), and the like.

The timing of adding the ROCK inhibitor, SHH (sonic hedgehog) signal transduction pathway-acting substance and/or FGF signal transduction pathway-acting substance is not particularly limited, and it is preferable to culture in a medium containing the ROCK inhibitor, SHH (sonic hedgehog) signal transduction pathway-acting substance and/or FGF signal transduction pathway-acting substance from the start of culture. These substances are preferably added to the medium at the same time, and more preferably added to the medium at the same time as the Wnt signaling pathway-acting substance. As one embodiment, the dispersed retinal cells may be cultured in a medium containing these substances for 1 to 14 days.

Suspension culture refers to culturing cells in a state of not adhering to a culture vessel, and is not particularly limited, and may be performed using the following culture vessel: a culture vessel which has not been subjected to artificial treatment (e.g., coating treatment with an extracellular matrix or the like) for improving adhesion to cells; or a culture vessel subjected to a treatment for artificially suppressing adhesion (for example, polyhydroxyethyl methacrylate (poly-HEMA), a coating treatment based on a nonionic surface active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (for example, a water-soluble polymer (Lipidure) having 2-methacryloyloxyethyl phosphorylcholine as a structural unit).

Suspension culture is performed, for example, using SFEB (Serum-free suspension culture of embryoid body-like aggregates of Serum-free cells of Serum-free Floatingculture of Embryoid Bodies-LIKE AGGREGATES) or SFEBq (WO 2005/12390) using dispersed retinal cells as starting cells.

The adherent culture is to culture cells in a state of adhering to a culture vessel, and is not particularly limited, and may be performed using a culture vessel or the like which has been subjected to an artificial treatment in order to improve adhesion to cells. The adhesion culture is preferably performed using a culture vessel or the like coated with an extracellular matrix and/or a temperature-responsive polymer. A method for producing retinal tissue according to one embodiment includes the steps of: the culture vessel coated with the temperature-responsive polymer is exposed to a temperature at which the properties of the temperature-responsive polymer change, thereby allowing the sheet-like retinal tissue to be peeled from the culture vessel.

The following sheet-like retinal tissue can be produced by performing an adhesion culture on a culture vessel coated with an extracellular matrix.

By culturing in the presence of an extracellular matrix, the cells recognize the basal membrane side, and therefore the apical surface is easily formed, and furthermore, the cells are oriented in a direction substantially perpendicular to the layer direction, so that a retinal tissue having a better layer structure can be obtained. By using a culture vessel or the like coated with a temperature-responsive polymer, the formed retinal tissue having an epithelial structure (or a multilayer structure) can be easily peeled off from the culture vessel or the like only by a temperature change, and no enzyme treatment is required, so that a strong sheet-like retinal tissue can be recovered without weakening the intercellular junction by the enzyme treatment. Therefore, it is preferable to use a culture vessel or the like coated with an extracellular matrix and/or a temperature-responsive polymer, in particular, both the extracellular matrix and the temperature-responsive polymer, and examples thereof include a culture surface of the culture vessel coated with the temperature-responsive polymer and an upper surface of the polymer coated with the extracellular matrix. The culture surface refers to the surface to which cells in the culture vessel adhere, and the upper surface of the polymer refers to the surface of the polymer coating opposite to the surface in contact with the culture surface.

The extracellular matrix refers to a biopolymer constituting the extracellular space, and includes cell-adhesive proteins such as fibronectin, vitronectin, and laminin, fibrous proteins such as collagen and elastin, fragments of these proteins, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, proteoglycans, matrigel, and the like. The extracellular matrix is preferably 1 or more selected from the group consisting of collagen, laminin, fibronectin, matrigel, vitronectin, and fragments of these proteins. Examples of the laminin fragment include commercially available products such as iMatrix-511, iMatrix-411, and iMatrix-221.

Matrigel is a basement membrane preparation derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared by the method disclosed in, for example, US patent No.4829000, and commercially available products can be purchased. The matrix gel comprises laminin, type IV collagen, heparan sulfate proteoglycan and nidogen.

The temperature responsive polymer is a polymer whose properties change according to a temperature change. That is, it has a lower critical solution temperature (Lower Critical Solution temperature, LCST) in water, and exhibits the following phase change behavior with a certain temperature as a critical point: at temperatures above this temperature, the intramolecular or intermolecular hydrophobic bonds are enhanced and the polymer chains aggregate, whereas at temperatures below this temperature, the polymer chains bind water molecules and hydrate. Specifically, for example, a temperature responsive polymer which maintains the hydrophobic state of the surface of the material at the culture temperature (about 37 ℃) and becomes hydrophilic at a temperature lower than the culture temperature, for example, about 20 to 30 ℃ and easily peels off the cultured cells can be mentioned. Therefore, the cells can be recovered as a single large sheet without requiring an enzyme treatment at the time of cell separation and without damaging the proteins between cells in the cells by the enzyme treatment. As such a temperature responsive polymer, for example, poly-N-isopropylacrylamide (PIPAAm) (LCST: 32 ℃ C.) is preferably used. In general, cell culture is carried out at about 37℃and if damage to cells due to low temperature is also considered, a temperature responsive polymer having an LCST in the range of about 20℃to 35℃is preferred. As a commercially available product, a temperature responsive cell culture apparatus for recovering a cell sheet having a temperature responsive polymer immobilized on the surface thereof (UpCell (registered trademark)) and the like are also available.

The culture conditions are not particularly limited, and the culture temperature is about 30 to 40 ℃, preferably about 37 ℃, and the concentration of CO ₂ is preferably about 2 to 5% when the culture is performed under an air atmosphere containing CO ₂.

Wnt signaling pathway-acting substances are necessary for the formation of layer structures in sheet retinal tissue, in particular the formation of the apical/basal plane (polarity of the apical/basal).

The concentration of the Wnt signaling pathway-acting substance may be a concentration capable of inducing a lamellar retinal tissue having a lamellar structure and a distal end surface. When CHIR99021 is used, the concentration of the Wnt signaling pathway-acting substance may be, for example, 0.01 μm to 100 μm, preferably 0.1 μm to 10 μm, and more preferably 1 μm to 10 μm. When another Wnt signaling pathway-acting substance is used, the concentration may be one that shows Wnt signaling activation to the same extent as CHIR99021 at the above concentration. Regarding whether or not a layer structure is formed, a person skilled in the art can easily determine by, for example, observation under a microscope, and measurement of thickness by an OCT or other device. The presence or absence of the formation of the apical surface can be confirmed by, for example, staining with an anti-Zo-1 antibody, an anti-ezrin antibody, or an anti-atypical PKC antibody.

The timing of adding the Wnt signaling pathway-acting substance is not particularly limited, and is preferably added as early as possible after the start of the adhesion culture. In one embodiment, the culture is preferably performed in a medium containing a Wnt signaling pathway-acting substance from the start of the culture. The number of days of culture in the medium containing the Wnt signaling pathway-acting substance is not particularly limited as long as the effect of reforming the multilayered structure (or multilayered structure) having the polarity of the apical surface/basal membrane can be observed, and is, for example, 1 to 15 days, preferably 1 to 9 days.

The culture may be continued in a medium from which Wnt signaling pathway-acting substances and the like have been removed. By continuous culture, the layer structure becomes thicker and differentiation of cells of the retinal lineage proceeds. The culture time is not particularly limited as long as it is a time until the inoculated dispersed retinal cell population proliferates and forms at least an epithelial structure (or a multilayer structure), and it is possible to culture a sheet-like retinal tissue until a target differentiation stage is produced. From the viewpoint of forming an epithelial structure (or a multilayer structure), it is desirable to culture for at least 7 days. The cultivation time may be, for example, 7 days to 60 days or less, and may be 40 days or less, 30 days or less, 20 days or less, or 16 days or less (for example, 16 days).

After the formation of the epithelial structure (or multilayer structure), the culture may be further continued for the purpose of proliferating, differentiating or maturing the cells. The medium used in the further culture may or may not contain Wnt signaling pathway-acting substances, ROCK inhibitors, SHH (sonic hedgehog) signaling pathway-acting substances and/or integrin signaling pathway-acting substances.

The "multilayer structure" refers to a structure in which, in a polar (that is, having an epithelial structure) tissue having a basal membrane side and a distal end side, cell layers in which 2 or more layers of cells are arranged in the same direction are superimposed, and the tangential directions of the surfaces of the different layers are substantially parallel to each other. The multilayer structure preferably has polarities of a basal plane and a apical plane, and in one embodiment, the retinal tissue having the multilayer structure may be a sheet-like retinal tissue, and the cell aggregate containing the retinal tissue having the multilayer structure may be a sheet-like cell aggregate. In addition, in one embodiment, the cell layer may be a neural retinal precursor cell layer, ganglion cell layer, or visual cell layer.

In one embodiment of the present invention, in the multilayer structure, the orientation of the cells may be a direction substantially perpendicular to the layer direction. Here, "orientation of cells" means the shape of the nucleus and the orientation of the cell body extending along the basal membrane side and the apical side. The direction substantially perpendicular to the layer direction means a direction orthogonal to a direction in which cells of the layers in the multilayer structure are in contact and aligned (i.e., a tangential direction of the layer surface), and means a direction perpendicular to the layer or a longitudinal direction.

In one embodiment of the present invention, the method may further include the steps of: retinal tissue (particularly, sheet-like retinal tissue) having an epithelial structure (or a multilayer structure) obtained by suspension culture or adhesion culture is cut into a size required for transplantation. For example, cutting may be performed using forceps, knives, scissors, etc.

On the other hand, the following problems were found: when a sheet-like retinal tissue is produced by the above method, RPE cells are produced and mixed in a certain ratio. Although these RPE cells can be visually confirmed and removed, it is preferable that they are not included from the beginning from the viewpoint of quality and production efficiency. Accordingly, intensive studies have been made to solve the problem, and as a result, the incorporation of RPE cells can be significantly reduced by using the method disclosed below.

[ Sheet-like retinal tissue ]

One embodiment of the present invention is a sheet-like retinal (neural retinal) tissue having an epithelial structure. In one embodiment of the sheet-like retinal tissue, the sheet-like retinal tissue comprises a retinal cell layer having a multilayer structure with polarities of a basal plane and a apical plane, wherein the retinal cell layer having a multilayer structure contains 1 or more cells selected from the group consisting of retinal precursor cells, optic cell precursor cells and optic cells, and the orientation of the cells in the retinal cell layer is a direction substantially perpendicular to the layer direction. The term "sheet-like" as used herein refers to a structure having a single layer or a plurality of layers of cells that are biologically bound in at least two dimensions.

Any size of sheet-like retinal tissue can be produced depending on the culture device used, which is one of the advantages of the present invention. That is, it is possible to produce a sheet-like retinal tissue of a size which has not been produced conventionally, and when the disease is spread over a wide range, it is possible to treat the disease by transplanting 1 sheet.

In one embodiment, the sheet-like retinal tissue of the present invention has a major axis (also referred to as a diameter) of, for example, 2mm or more, 4mm or more, 5mm or more, 7.5mm or more, or 10mm or more. In one embodiment, the sheet-like retinal tissue of the present invention has a minor diameter of, for example, 2mm or more, 3mm or more, 4mm or more, or 5mm or more. In principle, there is no upper limit on the major axis and the minor axis, but it is limited by the size of the culture dish or the like used for the culture. As one embodiment, the long diameter may be 10cm or less, 5cm or less, 4cm or less, 3cm or less, 2cm or less, or 1cm or less. In one embodiment, the height of the sheet-like retinal tissue of the present invention may be, for example, 50 μm to 1500 μm, preferably 200 μm to 700 μm.

The method for measuring the long diameter, short diameter and height of the sheet-like retinal tissue is not particularly limited, and can be measured from an image captured under a microscope, for example. For example, regarding a sheet-like retinal tissue, a front image, which is taken with the tip end facing toward the objective lens side, and a lateral image, which is taken in an inclined state so as to be perpendicular when viewing a section from an objective lens, can be taken with a stereoscopic microscope and measured from the taken images. Here, the major axis refers to the longest line segment and the length thereof among the line segments connecting the 2 end points on the sheet cross section in the front image. The minor axis refers to the longest line segment and the length thereof among the line segments orthogonal to the major axis among the line segments connecting the 2 end points on the sheet cross section in the frontal image. The height refers to the longest line segment and the length thereof among line segments orthogonal to the segment cross section and ending with the intersection point with the segment cross section and the vertex of the retinal sheet.

The basal surface and the apical surface are as defined above. By "the multilayer structure has a polarity of the basal plane and the apical plane" is meant that the basal plane is present on one side of the multilayer structure and the apical plane is present on the other side. A base film may be present on the base surface.

"The direction of the cells is a direction substantially perpendicular to the layer direction" means a direction in which the long diameters of the cells present in each layer of the retina-based cell layer are oriented substantially perpendicular to the layer direction. The substantially vertical direction means that an acute angle formed by the layer direction and the long diameter of the cell is about 75 ° (or 80 °) to 90 ° or less. In the present specification, if about 50% or more, preferably 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more of the cells present in each layer are oriented in a direction substantially perpendicular to the layer direction, it is determined that "the orientation of the cells is a direction substantially perpendicular to the layer direction".

The sheet retinal tissue is preferably free of RPE cells. In one embodiment, the ratio of the number of RPE cells to the total number of cells in the sheet-like retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Regarding the ratio of the number of RPE cells to the total number of cells in the sheet-like retinal tissue, the ratio of cells expressing the markers of the above RPE cells can be measured, for example, using flow cytometry (FACS) or the like. In one embodiment, the proportion of the RPE cell area in the total area of the sheet-like retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The RPE cells are black, so the proportion of the area of the RPE cells in the total area of the sheet-like retinal tissue can be calculated from the proportion of the area that is black under the microscope. Alternatively, cells expressing markers of RPE cells may be detected by PCR or the like. The method of reducing the proportion of RPE cells in the sheet-like retinal (neural retinal) tissue is as described above.

In a sheet-like retinal tissue composed of a retinal cell layer having a multilayer structure according to one embodiment,

(4) The diameter is more than 8 mm.

(Sheet-like retinal cell layer (neural retina) -retinal pigment epithelial cell complex (composite sheet))

As one embodiment of the present invention, the retinal cell-based layer having a multilayer structure may further contain a sheet-like retinal pigment epithelial cell bonded to the retinal cell-based layer, and examples thereof include a complex (a compact sheet) in which the tangential directions of the surfaces of the retinal cell-based layer and the sheet-like retinal pigment epithelial cell are substantially parallel, the distal end surface of the retinal cell-based layer and the distal end surface of the sheet-like retinal pigment epithelial cell face each other, and the retinal cell-based layer and the sheet-like retinal pigment epithelial cell are bonded by an adhesion factor existing between them. The sheet-like neural retina is also called a neural retina sheet or an NR sheet, and the sheet-like retinal pigment epithelial cells are also called a retinal pigment epithelial cell sheet or an RPE cell sheet (RPE sheet). That is, one embodiment of the present invention also provides a complex (a compact) of a neural retina and RPE cells. As another embodiment of the present invention, a complex (a complex sheet) in which scattered retinal pigment epithelial cells are bonded to the distal end surface side of a sheet-like retinal cell layer (a sheet-like neural retina) by an adhesion factor is also included.

In the complex of the present specification, the tangential directions of the neural retina sheet and the retinal pigment epithelial cell sheet are substantially parallel to each other. "substantially parallel to the tangential direction" means that the neural retina is parallel to the tangential direction of the mutually facing surfaces of the retinal pigment epithelial cell sheet. In the complex of the present invention, the tip surface of the neural retina and the tip surface of the retinal pigment epithelial cell face each other. That is, the distal end face of the neural retina and the distal end face of the retinal pigment epithelial cell exist in a state of being close to each other.

As described later, a complex (a complex sheet) of the neural retina and the RPE cells can be prepared by joining a sheet-like retinal cell-line layer (a sheet-like neural retina) with the RPE cell sheet or the dispersed RPE cells in the presence of an adhesion factor. The sheet-like retinal cell layer (sheet-like neural retina) can be produced by the above-described production method of a cell aggregate containing a retinal tissue having an epithelial structure (or a multilayer structure). The RPE cell sheet can be produced by a method for producing a retinal pigment epithelial cell sheet described later.

(Method for producing retinal pigment epithelial cell sheet)

The Retinal Pigment Epithelial (RPE) cells are derived from pluripotent stem cells, and specifically can be obtained by differentiation induction of pluripotent stem cells. Examples of the method for producing retinal pigment epithelial cells include the methods disclosed in WO2005/070011, WO2006/080952, WO 2011/063175, WO2012/173207, WO2015/053375, WO2015/053376, WO2015/068505, WO2017/043605, stem Cell Reports,2 (2), 205-218 (2014) and CELL STEM CELL,10 (6), 771-785 (2012), but are not particularly limited. Further, retinal Pigment Epithelium (RPE) cell sheets can be prepared by modifying the method described in WO 2016/063985. Retinal pigment epithelial cells can be produced in the form of cell sheets or spheroid cell aggregates. When the RPE cell sheet is produced in the form of spherical cell aggregates, the cell aggregates can be cut by, for example, forceps, knives, scissors, or the like.

As a modification of the method described in WO2016/063985, in the above method, pluripotent stem cells are 1) treated with a TGF-beta family signal transduction pathway inhibitor and a sonic hedgehog signal transduction pathway-acting substance one day before differentiation induction, and 2) cultured without treatment with a sonic hedgehog signal transduction pathway-acting substance at the beginning of differentiation induction. Thereafter, the above steps (B) and (C) are performed. Further, it is preferable to advance the start timing of the step (D). Specifically, step (D) is started about 9 days (for example, after 7 days, 8 days, 9 days, 10 days, 11 days) from the start of the suspension culture in step (B). And then the step (E) is implemented. By this method, spherical cell aggregates of RPE cells can be obtained. The cell aggregates may be dispersed to prepare a cell suspension, or RPE cell sheets may be prepared by cutting the cell aggregates with forceps, knives, scissors, or the like. RPE cell sheets may also be prepared by culturing a dispersed cell suspension by adherent culture. Further, by dispersing the RPE cell sheet or the aggregate of RPE cells, the dispersed RPE cells can be obtained.

The retinal pigment epithelial cell sheet may be further cultured to have a polygonal or paver-like cell morphology prior to contact with the cell aggregates of the neural retina. The medium in this case is not particularly limited, and may be replaced with a maintenance medium for retinal pigment epithelial cells (hereinafter, also referred to as RPE maintenance medium) and further cultured. Thus, the melanin deposition cell population and the cell population having a polygonal flat shape adhered to the basal lamina can be observed more clearly. The culture in the RPE maintenance medium is not limited as long as it can form colonies that proliferate while maintaining the properties of retinal pigment epithelial cells, and for example, it may be cultured for 5 days or more (for example, about 5 to 20 days) while performing the entire medium exchange at a frequency of 1 or more times for 3 days. The culture time can be easily set by those skilled in the art while confirming the form thereof. For example, the medium described in IOVS, march2004, vol.45, no.3, masatoshiHaruta, etc., IOVS, november 2011, vol.52, no.12, okamoto, etc., CELLSCIENCE (17), fumitaka Osakadar, etc., ebruary 2008, vol.49, no.2, gamm, etc., can be used as the maintenance medium for retinal pigment epithelial cells.

The long diameter of the retinal pigment epithelial cell sheet may be the same as that of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the length of the retinal pigment epithelial cell sheet may be, for example, 3 to 50mm, 5 to 30mm, 10 to 20mm, or the like.

The minor diameter of the retinal pigment epithelial cell sheet may be the same as that of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the minor diameter of the retinal pigment epithelial cell sheet may be, for example, in the range of 2mm to 40mm, 5mm to 30mm, 10mm to 20mm, or the like.

The degree of melanin deposition of the retinal pigment epithelial cell sheet is not particularly limited. The degree of melanin deposition in the retinal pigment epithelial cells contained in the retinal pigment epithelial cell sheet is preferably equal between cells. As one embodiment, the average melanin content of the retinal pigment epithelial cell sheet may be less than 20 pg/cell, less than 15 pg/cell, less than 10 pg/cell, less than 8 pg/cell, less than 7 pg/cell, less than 6 pg/cell, less than 5 pg/cell, less than 4 pg/cell, less than 3 pg/cell, less than 2 pg/cell, less than 1 pg/cell. The average melanin content of the retinal pigment epithelial cell sheet may be 0.1 pg/cell or more, 0.5 pg/cell or more, 1 pg/cell or more, 2 pg/cell or more, or 5 pg/cell or more.

The melanin content in the retinal pigment epithelial cell sheet can be measured, for example, as follows: after the retinal pigment epithelial cell sheet is dispersed, measurement is performed using a spectrophotometer or the like using a cell extract obtained by extraction with NaOH or the like. The average melanin content can be obtained by dividing the melanin content by the total number of cells contained in the retinal pigment epithelial cell sheet.

The complexes described above may be produced by conjugating a neural retinal sheet to the dispersed RPE cells or RPE cell sheets. Preferably, the neural retina sheet is a composite sheet obtained by bonding an RPE cell sheet. The above-described neural retina sheets and RPE cell sheets can be easily removed from the culture device by using forceps, knives, scissors, or the like. The two kinds of sheets taken out may be transferred to a new container (culture device or the like), or one kind of sheet may be left in the culture device and the other kind of sheet may be transferred to the culture device. By manufacturing the sheet-like tissue with the same size, the size of the sheet-like tissue to be joined can be made uniform. In addition, when using the neural retinal sheet and the retinal pigment epithelial cell which are cultured for different days in the above production method as the neural retinal sheet and the retinal pigment epithelial cell which are in contact with each other, the date of starting production may be shifted.

The above-mentioned neural retinal sheet is preferably contacted with retinal pigment epithelial cells in the presence of an adhesion factor. The adhesion factor is a substance having an effect of adhering cells to each other, and is not particularly limited, and examples thereof include the extracellular matrix and artificial hydrogels described above. The adhesion factor need not be an isolated single substance, but also includes preparations from organisms or cells such as matrigel, intercellular matrix, serum, etc. Matrigel is a basement membrane preparation from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared by the method disclosed in US patent No.4829000, for example, and commercially available products can be purchased. The matrix gel comprises laminin, type IV collagen, heparan sulfate proteoglycan and nidogen. The intercellular matrix is a generic term for extracellular matrix present between cells of the retinal system such as the cells of the organism's retina, and includes, for example, hyaluronic acid. The intercellular matrix can be collected from the retina of an organism by, for example, subjecting the retina to swelling and separation in distilled water by those skilled in the art, and commercially available products can be purchased. As the adhesion factor, an extracellular matrix or a hydrogel is preferable. Further, the extracellular matrix is preferably 1 or two or more kinds selected from the group consisting of hyaluronic acid, fibrin, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. The hydrogel is preferably 1 or two or more hydrogels selected from the group consisting of gelatin, fibrin, collagen, pectin, hyaluronic acid, and alginic acid. There are also substances classified into both extracellular matrix and hydrogel, and in this specification, the substances are treated as hydrogel when used as gel-like adhesion factors. Examples of commercially available extracellular matrices include Corning (registered trademark) matrigel basement membrane matrices, iMatrix511, and the like. The adhesion factor may be 1 or more selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan and entactin, and particularly preferably gelatin or fibrin.

The fibrin gel refers to gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution. A commercially available BOLHEAL (registered trademark) tissue adhesive may also be used. The fibrinogen solution may be reacted by contacting or mixing the two with a thrombin solution.

"Gelatin" refers to a substance obtained by pre-treating water-insoluble collagen with, for example, an acid or alkali, and thermally hydrolyzing the collagen to solubilize the collagen. Commercially available gelatin may be used, and examples thereof include gelatin LS-H (New field gelatin Co., pig skin alkali-treated gelatin, non-heat-treated gelatin, high gel strength), gelatin LS-W (New field gelatin Co., pig skin alkali-treated gelatin, heat-treated gelatin, low gel strength). Alkali-treated (lime-treated) gelatin (type B gelatin) is preferred, with heat-treated gelatin being preferred.

For hydrogels, the solution changes phase from gel to sol and from sol to gel by heating or cooling. Hydrogels can be gelled (jelly) by cooling and losing fluidity, and can be sol (aqueous solution) by heating and obtaining fluidity. The "melting point" herein means a temperature at which gelation occurs under a certain pressure, and the "freezing point" means a temperature at which gelation occurs under a certain pressure. The hydrogel is preferably a hydrogel that is degradable in an organism. As one embodiment, a hydrogel (e.g., gelatin) having a melting point at a temperature in the vicinity of body temperature (25 ℃ C. To 40 ℃ C.). The melting point of the hydrogel in the present specification may be 20℃to 40℃such as 20℃to 35℃and 25℃to 35℃and 30℃to 40℃and 35℃to 40 ℃. Typically, the melting point of a gel is a measure of the strength of the network, and as the concentration and molecular weight of the hydrogel increases, the melting point of the hydrogel (e.g., gelatin) also increases. In addition, for example, there is a tendency that the melting point and the solidification point are raised when the solid content is increased by saccharides. The melting and solidification points can thus be varied within a certain range. The method for measuring the melting point of the hydrogel is not particularly limited, and can be measured by a method specified in JIS K6503, for example.

The strength of the hydrogel is only required to be such an extent that the hydrogel does not disintegrate in the procedure for implantation. As an index of the strength of the hydrogel, there is "gel strength". The "gel strength" of a hydrogel refers to the mechanical strength of the object forming the gel. Usually expressed in terms of the force required to deform a shaped gel or the force required to break the gel (units: g, dyne (s)/cm ² or g/cm ²), primarily on the scale of the gel's stiffness. 1 dyne (s)) is defined as a force that, when applied to an object of 1 gram (g), provides an acceleration of 1 centimeter per second (cm/s ²) along its direction. For example, the gel strength of gelatin can be measured by a method specified in JIS K6503. In the present specification, for example, the gel strength of a hydrogel (e.g., gelatin) gel may be 50g or more, 100g or more, 200g or more, 500g or more, 1000g or more, 1200g or more, 1300g or more, 1400g or more, or 1500g or more. The gel strength of the hydrogel (gelatin) may be 3000g or less, 2500g or less, or 2000g or less.

The concentration of the adhesion factor (extracellular matrix) varies depending on the size of the neural retinal sheet or retinal pigment epithelial cell sheet and the number of cells of the retinal pigment epithelial cells, and can be easily set by a person skilled in the art by confirming the adhesion state of RPE cells. For example, in the case of matrigel, it is preferable to add it in a concentration of 200 to 10000 times diluted with a conventional product (Corning Co.), and in the case of iMatrix, it is preferable to add it in a concentration of 0.1 to 5. Mu.g/mL.

As one embodiment of the method for producing the complex using an extracellular matrix, culture for adhering a neural retinal sheet to retinal pigment epithelial cells or retinal pigment epithelial cell sheets may be performed in a medium containing an adhesion factor (extracellular matrix). The medium to be used is not particularly limited, and examples thereof include a medium used in culturing retinal pigment epithelial cells or neural retinas (e.g., DMEM/F12 medium, neurobasal medium, a mixed medium of these, RPE maintenance medium, etc.). In addition, the culture for adhesion may be performed in the presence of other components such as growth factors such as EGF together with the extracellular matrix. The culture may be continued in the medium containing the adhesion factor for a certain period of time (for example, 1 to 10 days) for the culture to adhere the neural retinal sheet to the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet, or may be continued after the culture in the medium containing the adhesion factor is exchanged for a medium containing no adhesion factor.

The neural retinal sheet or retinal pigment epithelial cells or retinal pigment epithelial cell sheet may be coated with an adhesion factor before the culture for adhering the neural retinal sheet to the retinal pigment epithelial cells or retinal pigment epithelial cell sheet is performed. Specifically, the neural retinal sheet or retinal pigment epithelial cell sheet may be cultured in the medium containing the adhesion factor. The incubation time can be appropriately set by those skilled in the art, and may be about 10 minutes to 5 hours (for example, about 10 minutes to 60 minutes). After incubation, washing may be performed with a medium such as PBS.

The adhesion factor, hydrogel or matrigel is preferably a fibrin gel. The fibrin gel is a gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution. In the present specification, a substance having a property of forming a gel by a reaction is referred to as a matrix precursor, and thrombin and fibrinogen, for example, which form a fibrin gel by a reaction, are examples of the matrix precursor. The fibrinogen solution can be prepared by dissolving fibrinogen powder or the like in a fibrinogen-soluble solution containing aprotinin polypeptide, and the concentration thereof is not particularly limited, and is, for example, 40 to 480mg/mL, preferably 80 to 320mg/mL (for example, 160 mg/mL). When the activity of factor VIII contained in 1mL of normal human plasma is 1 unit, the concentration may be 37.5 units/mL to 225 units/mL (for example, 75 units/mL). The thrombin solution can be prepared by dissolving thrombin powder or the like in a thrombin solution containing calcium chloride hydrate, and the concentration thereof is not particularly limited, and is, for example, 125 units/mL to 750 units/mL (for example, 75 units/mL). The fibrinogen solution and the thrombin solution may be reacted by contacting or mixing them, and in this case, the ratio of activity is preferably 1:1 to 1: 9. preferably 1: 3-1: 4, a fibrinogen solution and a thrombin solution are used.

As one embodiment of the method for producing the composite using the hydrogel, a method for producing the composite using the fibrin gel is described below. Specifically, a neural retinal sheet is adhered to retinal pigment epithelial cells or retinal pigment epithelial cell sheets by a fibrin gel produced by reacting fibrinogen with thrombin.

In one embodiment, the fibrinogen and thrombin are reacted to gel by bringing 1 tissue (retinas or retinal pigment epithelial cells) of the 2 tissues after contact with the fibrinogen solution and the other 1 tissue (retinas or retinas) after contact with the thrombin solution into contact with each other. Here, contact with the solution means: at least the adhesion surface of the tissue (the surface facing the other tissue when the fibrin gel is adhered to the other tissue) is brought into contact with the fibrinogen solution or the thrombin solution to such an extent that the solution adheres to the adhesion surface of the tissue.

More specifically, for example, when the neural retina sheet is adhered to the retinal pigment epithelial cell sheet, the neural retina sheet may be immersed in a fibrinogen solution, or the retinal pigment epithelial cell sheet may be immersed in a thrombin solution (the solution may be reversed). Preferably in terms of volume ratio 3:1 to 1:3 using fibrinogen solution and thrombin solution. Excess fibrinogen or thrombin attached to the tissue may be removed prior to adhesion. By this operation, the thickness of the fibrin gel can be adjusted.

In order to adhere the neural retina sheet after the fibrinogen solution and the retinal pigment epithelial cell sheet after the thrombin solution, both may be contacted. Here, contacting the tissue means that the surface to which the fibrinogen solution is attached and the surface to which the thrombin solution is attached are brought into contact, that is, overlapped. Further, it is preferable that the retinal pigment epithelial cell sheet and the neural retina are adhered so that the tangential directions of the surfaces of the two cells are substantially parallel and that the distal end surface of the neural retina and the distal end surface of the retinal pigment epithelial cell sheet face each other.

In one embodiment, the tissue may be contacted with a fibrinogen solution, and then a thrombin solution may be added to the fibrinogen solution to react fibrinogen with thrombin. In this case, the tissue is embedded in the fibrin gel.

In one embodiment, the method of manufacturing of the present invention may further include a step of cutting out the composite of a size required for transplantation from the composite. Since more than 2 tissues are firmly adhered by the fibrin gel, the graft can be easily cut to a desired size without peeling the tissues from the composite when cut out of the composite. Forceps, knives, scissors, etc. may be used for cutting.

[ Pharmaceutical composition, method of treatment, therapeutic agent and use ]

As one embodiment of the present invention, a pharmaceutical composition containing a sheet-like retinal tissue is exemplified. The pharmaceutical composition preferably contains, in addition to the sheet-like retinal tissue of the present invention, a pharmaceutically acceptable carrier. The pharmaceutical composition can be used for treating diseases based on the disorder of cells of the neural retinal lineage or the neural retina or damage to the neural retina. Examples of the diseases based on the neural retinal cell line or the neural retina disorder include ophthalmic diseases such as retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous choriocaulopathy, cone dystrophy, cone rod dystrophy, and the like. Examples of the damaged state of the neural retina include a state in which the retinal cells are denatured and dead.

As the pharmaceutically acceptable carrier, physiological aqueous solvents (physiological saline, buffer, serum-free medium, etc.) can be used. If necessary, the pharmaceutical composition may contain a preservative, a stabilizer, a reducing agent, an isotonizing agent, etc. which are generally used for a drug containing transplanted tissues or cells in transplantation medicine.

As one embodiment of the present invention, there is provided a therapeutic agent for a disease caused by a neural retinal disorder, which comprises the sheet-like retinal tissue obtained in the present invention. In addition, as one embodiment of the present invention, there is a method for treating a disease caused by a disorder of a neural retina cell or a neural retina or damage of the neural retina, which includes a step of transplanting the sheet-like retinal tissue obtained in the present invention to a subject in need of transplantation (for example, under the retina of an eye in which an ophthalmic disease has occurred). The sheet-like retinal tissue of the present invention can be used as a therapeutic agent for a disease caused by a disorder of the neural retina or to supplement the damaged portion in the damaged state of the neural retina. The disorder based on a neural retinal line cell or a neural retina or the damaged state of the neural retina can be treated by supplementing the neural retinal line cell or the neural retina where the disorder has occurred by transplanting the sheet-like retinal tissue of the present invention to a patient in need of transplantation having the disorder based on a neural retinal line cell or a neural retina or a patient in the damaged state of the neural retina. Examples of the transplanting method include a method of transplanting a sheet-like retinal tissue under the retina at a damaged portion by cutting the eyeball or the like. Examples of the implantation method include a method of implanting using a thin tube and a method of grasping the thin tube with forceps and implanting the thin tube, and examples of the thin tube include an injection needle.

As one embodiment of the present invention, there is provided the sheet-like retinal tissue obtained in the present invention for use in the treatment of diseases based on disorders of retinal lineage cells or retinal tissue or damage of retinal tissue. Further, as one embodiment of the present invention, there is provided a use of the sheet-like retinal tissue obtained in the present invention for producing a therapeutic agent for a disease caused by a disorder of retinal cells or retinal tissue or damage of retinal tissue.

Examples

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 exploration of aggregate reformation factors

For a composition having Rx: : human ES cells (KhES-1 strain (non-patent document 3)) and human iPS cells (1231 A3 strain, obtained from kyoto university) genetically modified with the Venus reporter gene were cultured under feeder-free conditions according to the method described in "SCIENTIFIC REPORTS,4,3594 (2014)". As feeder cells-free medium, stemFit medium (trade name: AK03N, manufactured by Weisu Co., ltd.) was used, and as a scaffold for replacing feeder cells, laminin 511-E8 (trade name, manufactured by Yu Co., ltd.) was used.

Specific maintenance culture operations of human ES and human iPS cells (human ES/iPS cells) were performed as follows. First, human ES/iPS cells which have reached sub-confluence (the extent of coverage of 6 cells in the culture area) were washed with PBS and then dispersed into single cells with TRYPLE SELECT (trade name, manufactured by Life Technologies). Here, the term "dispersed into single cells" means that the single cells are dispersed so that the single cells are formed, and the single cells may be contained in a cell group in which 2 to 50 cells are dispersed. Human ES cells dispersed into single cells were then seeded on plastic dishes coated with laminin 511-E8 and cultured in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10. Mu.M) in the absence of feeder cells. When a 6-well plate (i.e., a cell culture area 9.4cm ², manufactured by Kido corporation) is used as the plastic culture dish, the number of cells to be inoculated into the single-cell-dispersed human ES/iPS cells is 0.4 to 1.2X10. 10 ⁴ cells per 1 well. 1 day after inoculation, the medium was exchanged for StemFit without Y-27632. Thereafter, 1 to 2 days 1, medium exchange was performed with StemFit medium containing no Y-27632. Thereafter, culture was carried out in feeder cells-free conditions until the day before sub-confluence was reached. Human ES cells from the day before the sub-confluence were cultured in the presence of SB431542 (TGF-beta signaling pathway inhibitor, 5. Mu.M) and SAG (Shh signaling pathway inhibitor, 300 nM) (preconditioning treatment) for 1 day in the absence of feeder cells.

After washing the human ES/iPS cells with PBS, the cells were subjected to cell dispersion treatment with TRYPLE SELECT, and then dispersed into single cells by a blow operation, and then the single cell-dispersed human ES cells were suspended in 100. Mu.L of serum-free medium at 1.2X10. 10 ⁴ cells per 1 well in a non-cell-adhesive 96-well culture plate (trade name: primeSurface well V bottom plate, manufactured by Sumitomo electric Co., ltd.) and cultured in suspension at 37℃under 5% CO ₂. Serum-free medium (gfCDM +KSR) at this time was used to 1 of F-12 medium and IMDM medium: 1, a serum-free medium comprising 10% KSR, 450. Mu.M 1-thioglycerol and 1 Xa lipid concentrate having a clear chemical composition.

At the beginning of suspension culture (at the beginning of differentiation induction, day 0), Y-27632 (ROCK inhibitor, final concentration 10. Mu.M or 20. Mu.M) and SAG (Shh signaling pathway action substance, 300nM or 30nM or 0 nM) were added to the above serum-free medium. On day 3 after the initiation of suspension culture, 50. Mu.L of a medium containing no Y-27632 and SAG and exogenous human recombinant BMP4 (trade name: recombinant Human BMP-4, manufactured by R & D Systems Co.) at a final concentration of 1.5nM was used. Half-dose exchange was performed 1 time 3 days after day 6 from the start of suspension culture with medium without Y-27632 and SAG and human recombinant BMP 4.

For the cell aggregates derived from KhES-1, the aggregates from day 14 to day 18 after the start of the suspension culture were further transferred to a 90mm low adhesion culture dish (Petri dish 90. Phi. (deep type), manufactured by Sumitomo electric Co., ltd.) and cultured in a serum-free medium (medium obtained by adding 1% N2 supplement to DMEM/F12 medium) containing Wnt signal transduction pathway-acting substances (CHIR 99021, 3. Mu.M) and FGF signal transduction pathway-inhibiting substances (SU 5402, 5. Mu.M) at 37℃for 3 to 4 days under 5% CO ₂%. Thereafter, the cells were cultured in a low-adhesion culture dish (suspension culture dish 90. Phi. (deep type), manufactured by Sumitomo electric Co., ltd.) of 90mm for a long period of time with a serum medium (NucT medium) containing no Wnt signal transduction pathway-acting substance and no FGF signal transduction-pathway-inhibiting substance.

Aggregates on day 16 (from 1231A 3) or day 27 (from KhES-1) after the start of suspension culture were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37℃for 20-30 minutes, single cells were dispersed by blowing. These cells were suspended in 100. Mu.L of serum-free medium in a non-cell-adhesive 96-well culture plate (trade name: primeSurface-well V bottom plate, manufactured by Sumitomo electric Co., ltd.) so as to be 2X 10 ⁵ cells (from KhES-1) or 5X 10 ⁵ cells (from 1231A 3) per 1 well, and were cultured in suspension at 37℃under 5% CO ₂. At the same time as the addition of the cells, (1) no addition (control), (2) addition of 10. Mu.M or 20. Mu.M (from 1231A 3) Y-27632 (Wako Co.), (3) addition of 300nM SAG (Enzo Co.), (4) addition of 10. Mu.g/mL bFGF (Wako Co.), (5) addition of 1. Mu.M LDN193189 (Stemgent Co.), (6) addition of 3. Mu.M CHIR99021 (Wako Co.), (7) addition of 10. Mu.M SB431542 (Wako Co.), (8) addition of 3. Mu.M IWR-1 (Wako Co.).

When the cells were cultured for about 2 weeks under any of the conditions (1) to (8), the formation of aggregates was confirmed (see the results of example 3), and when Y-27632 was added, the formation of aggregates was confirmed to be newly formed by lifting from the 1 st to 2 nd days (1 st to 2 nd days) after the suspension culture for the formation of aggregates (FIG. 1: khES-1, FIG. 2:1231A 3). On the other hand, in the case of compounds other than Y-27632, the early aggregation reformation promoting effect was not seen (FIGS. 1 and 2). Therefore, it was found that by adding Y-27632 to retinal precursor cells dispersed into single cells, the reformation of aggregates can be promoted from an early stage.

Example 2 exploration of layer Structure formation promoting factors and aggregate proliferation promoting factors

For a composition having Rx: : human ES cells genetically modified with Venus reporter gene (KhES-1 strain (non-patent document 3)) were cultured under feeder cells-free conditions according to the method described in "SCIENTIFIC REPORTS,4, 3594 (2014)". As feeder cells-free medium, stemFit medium (trade name: AK03N, manufactured by Weisu Co., ltd.) was used, and as a scaffold for replacing feeder cells, laminin 511-E8 (trade name, manufactured by Yu Co., ltd.) was used.

Aggregates of day 20 (1231A 3) and day 27 (KhES-1) after initiation of suspension culture prepared as in example 1 were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. These cells were suspended in 100. Mu.L of a serum-free medium containing 10. Mu. M Y-27632 (Wako Co.) per 1 well in a 96-well culture plate (trade name: primeSurface-96 well V bottom plate, manufactured by Sumitomo electric Co., ltd.) having no cell adhesion, and cultured under the conditions of 37℃and 5% CO ₂. At the same time as the addition of the cells, (1) no addition (only 10. Mu. M Y-27632), (2) addition of 30nM SAG (Enzo Co.), 3 addition of 300nM SAG (Enzo Co.), 4 addition of 10ng/mL bFGF (Wako Co.), 5 addition of 1. Mu.M LDN193189 (Stemgent Co.), 6 addition of 3. Mu.M HIR99021 (Wako Co.), 7 addition of 10. Mu.M SB431542 (Wako Co.), 8 addition of 3. Mu.M IWR-1 (from KhES-1) or 10ng/mL EGF (from 1231A 3).

The state of the re-formation of aggregates was observed on days 1,7 and 14 after suspension culture for re-formation of aggregates using a bright field microscope and a fluorescence microscope (BZ-X810, manufactured by Keyence Co.). The results of KhES-1-derived cells are shown in FIGS. 3 to 5. In addition, the aggregate area was measured using Image J. The results of the KhES-1-derived cells are shown in FIG. 6 (average value obtained by measuring 12 cells). In addition, the results of observing the state of re-formation of aggregates on day 1 after suspension culture of cells derived from 1231A3 are shown in fig. 7. From FIGS. 3-7, it was confirmed that the group with SAG (300 nM) or CHIR99021 (3. Mu.M) added on the basis of Y-27632 reformed larger aggregates than the group with Y-27632 alone. The effect of SAG and CHIR99021 in promoting the proliferation and reformation of aggregates was confirmed.

Example 3 exploration of layer Structure formation promoting factors and aggregate proliferation promoting factors

Aggregates on day 26 after the start of suspension culture, which were prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. These cells were suspended in 100. Mu.L of serum-free medium at 3.0X10. 10 ⁴ cells per 1 well in a non-cell-adhesive 96-well culture plate (trade name: primeSurface-well V bottom plate, manufactured by Sumitomo electric Co., ltd.) and cultured in suspension at 37℃under 5% CO ₂. At the same time as the addition of the cells, (1) no addition (control), (2) addition of 10. Mu. M Y-27632 (Wako Co.), addition of 300nM SAG (Enzo Co.), addition of (3) 300nM SAG (Enzo Co.), addition of 10. Mu. M Y-27632 (Wako Co.), and addition of 300nM SAG (Enzo Co.), addition of (5) 3. Mu.M CHIR99021 (Wako Co.), addition of (6) 10. Mu. M Y-27632 (Wako Co.), addition of 3. Mu.M CHIR99021 (Wako Co.), addition of (7) 300nM SAG (Enzo Co.), addition of 3. Mu.M CHIR99021 (Wako Co.), addition of (8) 10. Mu.MY-27632 (Wako Co.), addition of 300nM SAG (Enzo Co.), and addition of 3. Mu.M CHIR99021 (Wako Co.).

The state of the re-formation of aggregates was observed on days 1, 14 and 28 (days 1, 14, 28) after suspension culture for re-formation of aggregates, that is, on days 27, 40 and 54 (dd 27, dd40, dd 54) after initiation of suspension culture for differentiation induction, and the obtained results are shown in fig. 8, 9 and 10. From fig. 8, 9, and 10, it was confirmed that aggregates were reformed when CHIR99021 was added. In addition, it was confirmed that aggregates were larger when SAG was added on the basis of CHIR 99021.

Frozen sections were prepared by fixing aggregates on day 15 (corresponding to day 41 of differentiation) and day 28 (corresponding to day 54 of differentiation) after suspension culture for re-forming aggregates with 4% pfa. For these frozen sections, immunostaining was performed with DAPI and Anti-Chx 10 antibodies (trade name: anti CHX10 antibodies, EXalpha Co.), anti-beta-catenin antibodies (R & D Systems Co.), anti-type IV collagen antibodies (Abcam Co.), anti-Zo-1 antibodies (Invitrogen Co.), anti-Ki 67 antibodies (BD Co.), anti-Pax 6 antibodies (BioLegend Co.), anti-RxR-gamma (RxRg) antibodies (Spring Bioscience Co.), anti-CRX antibodies (Abnova Co.), anti-NRL antibodies (R & D Systems Co.), anti-recovery protein antibodies (Proteintech Co.), anti-Islet-1 antibodies (DSHB Co.), anti-GS antibodies (Sigma Co.), anti-Brn 3 antibodies (Santa Cruz Co.), anti-calomen antibodies (R & D Systems Co.).

These immunostained sections were observed with a bright field microscope and a fluorescence microscope (BZ-X810, manufactured by Keyence Co., ltd.) and a confocal laser scanning fluorescence microscope (SP-8, manufactured by Leica Co., ltd.), and the results are shown in FIGS. 11 to 20. From fig. 11, 14, 15, and 18, it was confirmed that Rx-containing particles were formed on the outermost side of the aggregate when CHIR99021 was added: : layer structure of Venus positive, chx10 positive neural retinal precursor cells. In addition, according to fig. 12, 16 and 17, when CHIR99021 was added, a Zo-1 positive distal surface was formed on the outermost side of the aggregate, and a collagen positive basal surface was formed inside, and it was confirmed that an epithelial tissue having polarity of the distal end and the basal was formed. On the other hand, as shown in fig. 17 in particular, in the group to which CHIR99021 was not added, cell-free polarity was observed or a rose pattern structure was exhibited. Furthermore, from FIGS. 19 and 20, it was confirmed that cone precursor cells positive for RxR-. Gamma. (RxRg) and CRX and cone precursor cells positive for restorer protein and CRX were differentiated, and retinal ganglion cells positive for Islet-1 and Brn3 and calomelin-positive and non-long process cells were differentiated.

From these results, it was found that when CHIR99021 was added to a single-cell suspension of retinal cells which were temporarily dispersed, reorganization and retinal differentiation were performed to form retinal tissue which was a polar epithelial tissue having a basal lamina at the apex.

Example 4 differentiation period related to aggregate reformation

Aggregates on days 18, 25, 40, 61 and 75 (dd 18, dd25, dd40, dd61 and dd 75) after initiation of the suspension culture prepared as in example 1 were washed with PBS, and a nerve cell dispersion (WAKO Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. These cells were suspended in 100. Mu.L of serum-free medium containing 10. Mu. M Y-27632 (Wako Co.), 300nM SAG (Enzo Co.) and 3. Mu.M CHIR99021 (Wako Co.) in a 96-well culture plate (trade name: primeSurface-well V bottom plate, manufactured by Sumitomo electric Co.) with 2 to 5X 10 ⁵ cells per 1 well, and cultured in suspension at 37℃under 5% CO ₂.

The reformed state of the aggregates was observed on days 3, 15, and 21 (days 3, 15, and 21) of the culture for reforming the aggregates by a bright field microscope and a fluorescence microscope, and the obtained results are shown in FIG. 21. From fig. 21, it was confirmed that aggregates were newly formed and a layered structure was formed in the cell aggregates on any differentiation day of dd18, dd25, dd40, dd61, and dd 75. In addition, rx was observed: : venus positive retinal tissue characteristics were maintained (FIG. 21).

From these results, it was found that aggregates could be reformed by adding Y-27632 and SAG and CHIR99021 to retinal tissue at any stage of differentiation.

Example 5 aggregate reformation on brain organoids

On day 3 after the initiation of suspension culture, 50. Mu.L of a medium containing exogenous human recombinant BMP4 (trade name: recombinant Human BMP-4, manufactured by R & D Systems Co., ltd.) at a final concentration of 1.5nM without Y-27632 and SAG was used. In order to prepare brain organoids, a group to which BMP4 was not added was set. Half-dose exchange was performed 1 time 3 days after day 6 from the start of suspension culture with medium without Y-27632 and SAG and human recombinant BMP 4.

Aggregates on day 40 after the start of the suspension culture thus prepared were washed with PBS, and a neural cell dispersion (manufactured by WAKO Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. The dispersed cells were suspended in 100. Mu.L of serum-free medium containing 10mM Y-27632 (Wako Co., ltd.), 300nM SAG (Enzo Co., ltd.) and 3. Mu.M CHIR99021 (Wako Co., ltd.) at 37℃under 5% CO ₂ in a non-cell-adherent 96-well culture plate (trade name: primeSurface-well V bottom plate, manufactured by Sumitomo electric Co., ltd.) so as to be 5X 10 ⁵ cells per 1 well.

The state of the re-formation of the aggregates was observed on days 3, 15 and 21 of the culture for re-formation of the aggregates by a bright field microscope and a fluorescence microscope, and the results are shown in FIG. 22. According to fig. 22, confirm at Rx: : aggregates are also reformed and lamellar structures are formed in Venus-negative telencephaloids, as in retinal organoids.

From these results, it is found that aggregates can be reformed not only in retinal organoids but also in nerve epithelial tissues such as telencephaloids.

Example 6 cryopreservation Studies

For a composition having Rx: : human ES cells genetically modified with Venus reporter gene (KhES-1 strain (non-patent document 3)), were cultured under feeder cells-free conditions according to the method described in "SCIENTIFIC REPORTS, 3594 (2014)". As feeder cells-free medium, stemFit medium (trade name: AK03N, manufactured by Weisu Co., ltd.) was used, and as a scaffold for replacing feeder cells, laminin 511-E8 (trade name, manufactured by Yu Co., ltd.) was used.

Aggregates on day 31 after the start of suspension culture, which was prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing.

After dispersion, 100. Mu.L of serum-free medium containing 10. Mu. M Y-27632 (Wako Co., ltd.), 300nM SAG (Enzo Co.), and 3. Mu.M CHIR99021 (Wako Co., ltd.) was suspended in a 96-well culture plate (trade name: primeSurface well V bottom plate, manufactured by Sumitomo electric Co., ltd.) having no cell adhesion so as to be 2.0X10X 10 ⁵ cells per 1 well for the non-frozen group, and cultured under the conditions of 37℃and 5% CO ₂.

After the dispersion, the single cells were suspended in PBS (Gibco Co.) and the following frozen solutions, cell Banker (TaKaRa Co.), stem Cell Banker (TaKaRa Co.), cultureSure (registered trademark) frozen solution (CultureSure (registered trademark) frozen solution; fuji film and Wako pure chemical industries, ltd.), STEMdiff (trademark) Neural Progenitor frozen solution (Stemcell Co.), stemSure (registered trademark) frozen solution (Fuji film and Wako pure chemical industries, ltd.), bambanker (Japanese Genetics Co., ltd.) for negative control at 1.0X10. 10 ⁶ cells/mL, and frozen at-80℃using Fabricius' al.

After 1 day of cryopreservation, the viable cell rate was confirmed. The viable cell rate immediately after resuscitation was drastically decreased in the PBS group, but the viable cell rate was high in any of the frozen solutions, and the frozen solutions (Cell Banker, stem Cell Banker, cultureSure, stemSure, bambanker) other than STEMdiff showed no less than 90% of the frozen solutions (fig. 24 (a)).

These cells were suspended in 100. Mu.L of NucT0 medium containing 10. Mu. M Y-27632 (Wako Co., ltd.), 300nM SAG (Enzo Co., ltd.) and 3. Mu.M CHIR99021 (Wako Co., ltd.) in a 96-well culture plate (trade name: primeSurface well V bottom plate, manufactured by Sumitomo electric Co., ltd.) so as to have 2X 10/⁵ cells per 1 well, and cultured in suspension at 37℃under 5% CO ₂.

After 1 day of inoculation, it was confirmed that aggregates were formed again without freezing in any of the frozen stock solutions (Cell Banker 1, stem Cell Banker, cultureSure, STEMdiff, stemSure, bambanker) (fig. 23, 24 (B)). In particular Cell Banker 1, small Rx present in the peripheral portion of the aggregates observed in other frozen stock solutions was not confirmed: : venus positive cell aggregates gave a visual image close to non-frozen (FIG. 23).

It was found that the culture for reforming the aggregates was not inferior to the non-frozen culture in the case of any frozen stock solution after 7 days.

From these results, it was found that aggregates can be reformed by adding Y-27632, SAG and CHIR99021 after resuscitating and culturing the single-cell suspension of retinal-system cells obtained by temporary dispersion.

Example 7 scaffold protein for expression of retinal aggregates

As is clear from the studies in examples 1 to 6, it was found that the cells of the retina system were dispersed and the pluripotent stem cells were subjected to retinal differentiation to obtain cell aggregates containing the cells of the retina system, the cell aggregates were dispersed to obtain a single cell suspension, and Y-27632 (Wako Co.), SAG (Enzo Co.) and CHIR99021 (Wako Co.) were added to the single cell suspension to perform suspension culture, thereby allowing the reorganization of the retina tissue having a layered structure and a polarity. Then, as a study for producing a wide sheet-like retinal tissue, it is conceivable to conduct adhesion culture. And an extracellular matrix for performing adhesion culture was studied.

Frozen sections were prepared by fixing aggregates obtained 25 to 35 days after the start of suspension culture prepared as in example 1 with 4% pfa. For these frozen sections, immunostaining was performed with DAPI and anti-laminin antibodies (trade name: anti-laminin antibody, abcam Co.), anti-fibronectin antibodies (R & D systems Co.), anti-type IV collagen antibodies (Abcam Co.). These immunostained sections were observed with confocal laser microscopy. As a result, it was confirmed that the aggregate expressed at least laminin, fibronectin, and type IV collagen as the constituent of the basement membrane (fig. 25). That is, in this self-organized culture system, human retinal tissue automatically forms a basal membrane and expresses laminin, fibronectin, and type IV collagen.

The isoforms of laminin expressed in fetal mouse neural retinal tissue were investigated from well-known information on the staining of mouse embryonic tissue specialized as basement membrane (MOUSE BASEMENT MEMBRANE BODYMAP; http:// dbarcicedbc. Jp/architecture/matrixome/bm/home. Html). As a result, as the laminin isoforms, 1,4, and 5 are known for laminin α,1 and 2 for laminin β, and 1 for laminin γ (fig. 26).

The results suggest that, as an extracellular matrix suitable for neural retinal tissue, laminin a, laminin β, laminin γ combinations, laminin 511, 521, 411, 421, 111, 121 (particularly 511, 521, 411) may be useful.

That is, it has been suggested that laminin 511, laminin 521, laminin 411, fibronectin, type IV collagen, and the like may be useful as an extracellular matrix for reforming a sheet-like retinal sheet by adhesion culture.

Example 8 inoculation extracellular matrix study on Transwell >

In the studies of examples 1 to 6, a method of reforming aggregates containing retinal tissue having a layered structure from a single cell suspension of retinal cell lines by suspension culture was studied. In example 7, a candidate of the extracellular matrix used in the adhesion culture was studied. These combinations were studied for the method of reformation of flaky retinal tissue by adhesion culture from single cell suspensions of retinal lineage cells (re-flaking).

Aggregates on day 16 after the start of suspension culture, which was prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing.

As extracellular matrices that promote re-flaking, matrigel (Corning Co.) and laminin 511-E8 (trade name iMatrix, manufactured by Beta Corp.) were used. Single cells of the above-described dispersed retinal cells were suspended in 300. Mu.L of serum-free medium containing 10. Mu. M Y-27632 (Wako Co.), 300nM SAG (Enzo Co.), and 3. Mu.M CHIR99021 (Wako Co.) in the form of 0.5 to 4X 10 ⁵ cells per 1 well under the following 3 conditions and inoculated into 24-well Transwell (Corning Co.). After 3 days of inoculation, medium exchange was performed with serum free medium (gfCDM) without Y-27632, SAG and CHIR99021 for 3-4 days 1.

Condition 1: inoculating to 24-well Transwell (matrigel (precoated)) pre-coated with matrigel

Condition 2: inoculated into 24-well Transwell (iMatrix (precoated)) pre-coated with laminin 511-E8

Condition 3: in a 24-well Transwell without pre-coating, cells were inoculated with a culture broth supplemented with laminin 511-E8 (Mix method).

After 14 days of culture for re-formation of aggregates, cell aggregates (cell sheets) formed in a sheet form under either condition. The pellet-like cell aggregates were fixed with 4% pfa, and frozen sections were prepared. For these frozen sections, immunostaining was performed with DAPI and Anti-Chx 10 antibodies (ExAlpha Co.), anti-Zo-1 antibodies (trade name: anti Zo-1 Antibody, invitrogen Co.), anti-type IV collagen antibodies (Abcam Co.). These immunostained sections were observed using confocal laser scanning fluorescence microscopy. As a result, the presence of Chx 10-positive neural retinal precursor cells was confirmed in the matrigel (precoated), iMatrix (precoated), and Mix methods (fig. 27). Furthermore, zo-1 as a marker of the apical surface (the surface where the tight junction of the epithelial tissue was formed) was confirmed on the upper side (the side not in contact with the well) of the sheet-like cell aggregate and type IV collagen as a marker of the basal surface was confirmed on the lower side (the side in contact with the well) of the sheet-like cell aggregate under the conditions of matrigel (precoating) and iMatrix (precoating). It was confirmed that Rx with polarity of tip and base was reformed: : venus positive patches of retinal precursor cells. On the other hand, it was confirmed that polarity was not formed in the case of Mix method in which extracellular matrix was simultaneously administered.

From these results, it was found that, in the case of retinal cells (NR) dispersed into single cells, sheet-like retinal tissue having a tip-base polarity (re-flaking) can be produced again by coating laminin 511-E8 or matrigel in advance as a scaffold.

Example 9 confirmation of Effect of Vaccination on Transwell, re-flaking CHIR99021 >

Aggregates of the suspension culture prepared as in example 1 were washed with PBS 15 to 30 days after the start of the suspension culture, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, in a 12-well Transwell coated with laminin 511-E8, it was suspended in (1) 300. Mu.L of serum-free medium containing 10. Mu.M Y-27632 (Wako Co.), 300. Mu.L of serum-free medium containing 10. Mu.M Y-27632 (Wako Co.) and 300nM SAG (Enzo Co.), 3) 300. Mu.L of serum-free medium containing 10. Mu.M M Y-27632 (Wako Co.) and 5. Mu.M CHIR99021 (Wako Co.), and 4) 300. Mu.L of serum-free medium containing 10. Mu. M Y-27632 (Wako Co.), 300nM SAG (Enzo Co.), and 3. Mu.M CHIR99021 (Wako Co.), and the adherent culture was carried out at 37℃under 5% CO ₂. 3 days after inoculation, medium exchange was performed with serum-free medium (NucT 0) free of Y-27632, SAG and CHIR99021 for 3-4 days 1.

Cell viability was observed under all conditions as a result of observation of cell sheets obtained after 14 days of culture for re-formation of aggregates (fig. 29). Thus, these cell sheets were fixed with 4% pfa, and frozen sections were prepared. For these frozen sections, immunostaining was performed with DAPI and an Anti-Zo-1 antibody (trade name: anti-Zo-1 Antibody, manufactured by Invitrogen corporation), an Anti-type IV collagen antibody (Abcam corporation). These immunostained sections were observed using confocal laser scanning fluorescence microscopy. As a result, it was confirmed that Rx having polarity of the tip and the base was formed again with the addition of CHIR 99021: : venus positive patches of retinal precursor cells. On the other hand, it was confirmed that CHIR99021 was not added, the top end face was not constituted, and polarity was not formed (fig. 30).

From these results, it was found that, with respect to retinal cells (NR) dispersed as single cells, a retinal sheet having a tip-base polarity can be produced again by adding CHIR 99021.

EXAMPLE 10 investigation of the concentration and addition time of the inoculating and re-flaking CHIR99021 on Transwell >

Aggregates prepared as in example 1 were washed with PBS 33 days after the start of suspension culture (dd 33), and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, the cells were inoculated in 24-well Transwell coated with laminin 511-E8 in such a manner that 2 to 4X 10 ⁵ cells were contained per 1 well, and 300. Mu.L of serum-free medium containing 1. Mu.M, 3. Mu.M or 9. Mu.M of CHIR99021 (Wako Co.) in addition to 10. Mu. M Y-27632 (Wako Co.) and 300nM SAG (Enzo Co.) was added thereto, followed by adhesion culture at 37℃under 5% CO ₂ for 3 days (day 0 to day 3) or 6 days (day 0 to day 9). After 3 days of inoculation, medium exchange was performed 1 time 3 to 4 days.

After 14 days of culture for re-formation of aggregates (day 14, dd 47), the resulting cell sheets were observed, and as a result, survival was observed except for the group to which 6 days, 9 days CHIR99021 were added at 9 μm (fig. 31). Thus, these cell sheets were fixed with 4% pfa, and frozen sections were prepared. For these frozen sections, immunostaining was performed with DAPI. It was confirmed that thicker Rx was reformed by adding 3 to 9 days of CHIR99021 at 1 to 9. Mu.M: : venus positive patches of retinal precursor cells (FIG. 32).

From these results, it was found that retinal cells (NR) dispersed as single cells can be prepared again by adding CHIR99021 for 3 to 9 days at least at 1 to 9. Mu.M.

Example 11 study of scaffolds at the time of Vaccination on Transwell >

Aggregates prepared as in example 1 were washed with PBS 24 days after the start of suspension culture, and a nerve cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, the adhesive was applied to a well (24. Mu.L) of a membrane in which (1) no coating (Non Coat) and (2) 2. Mu.L of iMatrix (trade name, manufactured by Kogyo Co.), 2. Mu.L of iMatrix (trade name, manufactured by Koshi Co.), 2. Mu.L of iMatrix (trade name, manufactured by Kogyo Co.), 5 (5) 2. Mu.L of vitronectin (VTN-N) human recombinant protein (Thermo Co.), 6) 2. Mu.L of CELL START (Thermo Co.), 7) 5. Mu.L of laminin 111, 5. Mu.L of laminin 211, 5. Mu.L of laminin 221, 5. Mu.L of laminin 411, 5. Mu.L of laminin 511, 5. Mu.L of laminin 521 (Lamia Co.), 8) 5. Mu.L of laminin 332, (2. Mu.L of Corning matrix), inoculated in such a manner that 2.0X10. ⁵ cells were contained in each 1 well, and 200. Mu.L of serum-free medium containing 3. Mu.M of CHIR99021 (Wako Co.), 100. Mu.g/mL of FGF8 (Wako Co.) in addition to 10. Mu. M Y-27632 (Wako Co.) and 300nM SAG (Enzo Co.) was added thereto, followed by adhesion culture at 37℃for 3 days under 5% CO ₂. After 3 days of inoculation, medium exchange was performed 1 time 3 to 4 days.

The obtained cell sheets were observed 22 days after inoculation, and as a result, good neural retinal sheets were obtained in the case of scaffolds other than CELL START (fig. 33).

From these results, it was found that not only iMatrix and matrigel were aggregates (NR) dispersed in single cells, but also retinal sheets were able to be produced again for various scaffold proteins.

Example 12 inoculation on Transwell, re-flaking, confirmation of retinal differentiation >

For a composition having Crx: : human ES cells genetically modified with Venus reporter gene (KhES-1 strain (non-patent document 3)), were cultured under feeder cells-free conditions according to the method described in "SCIENTIFIC REPORTS, 3594 (2014)". As feeder cells-free medium, stemFit medium (trade name: AK03N, manufactured by Weisu Co., ltd.) was used, and as a scaffold for replacing feeder cells, laminin 511-E8 (trade name, manufactured by Yu Co., ltd.) was used.

Aggregates on day 15 after the start of suspension culture, which were prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, in a 24-well Transwell coated with laminin 511-E8, 2.0X10 ⁵ cells per 1 well were suspended in 200. Mu.L serum-free medium containing 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR99021 and incubated at 37℃for 3 days under 5% CO ₂. 3 days after inoculation, medium exchange was performed with serum-free medium (NucT 0) free of Y-27632, SAG and CHIR99021 for 3-4 days 1.

The resulting cell sheets were observed 44 days after inoculation (day 44, dd 59), and differentiation of Crx was observed as a result: : venus positive visual cells (FIG. 34).

Further, aggregates on day 29 after the start of suspension culture were used, and the same was used for re-flaking. Cell sheets on day 28 (day 67) of the reorganization culture were fixed with 4% pfa, and frozen sections were prepared. For these frozen sections, immunostaining was performed with DAPI and Anti-Ki 67 antibodies (R & D Systems Co.), anti-Chx 10 antibodies (trade name: anti CHX10 Anti-body, EX alpha Co.), anti-Pax 6 antibodies (BD Pharmingen Co.), anti-Brn 3 antibodies (Santa Cruz Co.), anti-RxR-gamma (RxRg) antibodies (Spring Bioscience Co.), anti-Crx antibodies (abnova Co.). As a control, 3D Retina (cell aggregates) of dd70, which was not planarized (re-flaked), was also stained (fig. 35 to 37).

After the cell sheet on day 57 was fixed with 4% pfa, whole immunostaining was performed without preparing a section. Immunostaining was performed with DAPI and Anti-Chx 10 antibodies (trade name: anti CHX10 antibodies, EX alpha Co.), anti-Sox 2 antibodies (BD Pharmingen Co.), anti-Ki 67 antibodies (BD Co.), anti-Pax 6 antibodies (BioLegend Co.), anti-Brn 3 antibodies (Santa Cruz Co.), anti-TUJ 1 antibodies (Millipore Co.), anti-Islet-1 antibodies (R & D Systems Co.), anti-Crx antibodies (abnova Co.), anti-restorer antibodies (Proteintech Co.), anti-Zo-1 antibodies (Invitrogen Co.), anti-type IV collagen antibodies (Abcam Co.).

As a result, chx 10-, pax 6-and Ki 67-positive retinal precursor cells, brn 3-positive retinal ganglion cells, rxRg-positive, crx: : venus-positive and Crx-positive cone precursor cells differentiated as compared to the control 3D Retina. Further, as a result of observation of the positional relationship with the Transwell under transmitted light, it was confirmed that retinal sheets were formed on the Transwell, and retinal ganglion cells that were strongly positive for Pax6 and negative for Chx10, which were locally present on the basal side, were locally present on the Transwell side (fig. 38). Further, the whole was observed by a confocal microscope without producing a slice, and as a result, it was confirmed that the cells had the polarity of the tip and the base, and various retinal cells were differentiated according to the polarity (fig. 39, 40, and 41).

Further, long-term culture was performed, and the cell sheet of dd112 was immunostained in the form of a sheet without producing a section. Staining with Anti-Ribeye antibodies (CtBP 2, BD company) and Anti-restorer antibodies (Proteintech company), anti-Pax 6 antibodies (BioLegend company) and Anti-Chx 10 antibodies (trade name: anti CHX10 Antibody, santaCruz company) resulted in Crx being observed in the same cell: : expression Ribeye was confirmed by expression of Venus and recovery protein (fig. 42). From this, it was confirmed that the visual cells differentiated from the re-flaked retina were matured to the extent that synaptorin was expressed. In addition, confirm, in Crx: : the state where Venus is locally present on the distal side and Chx10 and Pax6 are locally present on the inner side forms a patch (fig. 43).

From these results, differentiation of retinal cells was confirmed in retinal sheets reorganized on the Transwell, and maintenance of polarity was observed.

Example 13 uses Rx: : venus strain maintenance culture of retinal cell lines

Aggregates on day 31 after the start of suspension culture, which was prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, (1) control, (2) 20ng/mL FGF2, (3) 20ng/mL EGF, (4) 100nM SAG, (5) 1 unit LIF、(6)10ng/mL IGF-1、(7)100ng/mL PDGF-AA、(8)100ng/mL PDGF-AB、(9)10μg/mL GDNF、(10)20μg/mL BDNF、(11)2μM Pyrintegrin、(12)1μM BMP4, were incubated at 37℃under 5% CO ₂ for 3 days in a medium containing 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR99021 in 500. Mu.L of serum-free DMEM/F12 in a 12-well Transwell coated with 10. Mu.L of laminin 511-E8 in a manner of 2.0X10X ⁵ cells per 1 well. 3 days after inoculation, medium exchange was performed with serum-free medium (N2 added to DMEM/F12) free of Y-27632, SAG and CHIR99021 for 3-4 days 1.

After 34 days, the cell sheets were fixed with 4% pfa and nuclear stained with DAPI. Looking at Rx: : area of Venus positive cells was observed, and the maintenance state of retinal lineage cells was studied. Observations were made using a fluorescence microscope (BZ-X810, manufactured by Keyence Co., ltd.) to confirm that Rx when EGF and FGF2 were continuously added at 20 ng/mL: : venus positive cells were widely maintained (FIG. 44).

From this, it can be seen that if EGF and FGF are added during the reorganization of retina, rx is maintained: : venus positive cells are effective.

Example 14 maintenance culture of retinal precursor cells at the time of planarization culture

Aggregates (NR) on day 27 after the start of suspension culture prepared in example 1 were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, cells were inoculated in a 12-well Transwell coated with 10. Mu.L of laminin 511-E8, suspended in less than 500. Mu.L of medium containing 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR99021 in 8.0X10 ⁵ cells/well. To study the medium in which retinal precursor cells were maintained, 20ng/mLFGF2 and 20ng/mLEGF were added to the medium of (1) NeuroCult NS-A (STEMCELL Technologies Co.), (2) STEMdiff Neural Progneitor (STEMCELL Technologies Co.), (3) StemPro NSC SFM (Thermo Fisher Co.), (4) RHB-A (TaKaRase:Sub>A Co.) and cultured for 1 month. As a control, nucT media was set. After 3 days of inoculation, medium exchange was performed with medium free of Y-27632, SAG and CHIR99021 for 3-4 days 1.

After 16 days of culture for re-formation of aggregates, the cell precursor cell markers Crx were visualized: : venus was examined for non-differentiation conditions (Crx:: venus does not emit light when observed with ase:Sub>A fluorescence microscope), and it was confirmed that NeuroCult NS-A and RHB-A mediase:Sub>A had some effect of delaying differentiation (FIG. 45).

From this, it was found that the addition of NeuroCult NS-A and RHB-A mediase:Sub>A and EGF, FGF had the effect of maintaining retinal precursor cells and delaying differentiation during the re-flaking.

Example 15 culture on collagen gel

Collagen gel was prepared using beMatrix low endotoxin collagenous solution (neo-field gelatin company). As a specific procedure, collagen AT was mixed with 5xDME and a buffer for reconstitution AT 7:2:1, coated onto a Transwell mesh, and incubated in an incubator at 37℃for 30 minutes with 5% CO ₂. After incubation, medium was added to the inside and outside of the inserts.

Aggregates (NR) on day 26 after the start of suspension culture prepared in example 1 were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, cells were seeded at 8.0X10 ⁵ cells/well on a 12-well Transwell collagen gel. The initial 3 days were incubated in the presence of 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR 99021. After 3 days of inoculation, medium exchange was performed with serum-free medium (NucT medium) without Y-27632, SAG and CHIR99021 for 3-4 days 1.

After 33 days of incubation for re-formation of aggregates, stereoscopic microscopy was used at Rx: : venus as an indicator, and as a result, it was confirmed that Rx was present in front on the Transwell collagen gel: : venus positive cells (FIG. 46).

From this, it was found that the collagen gel could be re-flaked by adding Y-27632 and SAG and CHIR 99021.

Example 16 transplantation study

Day 26 aggregates (NR) on the collagen gel prepared as in example 15 were washed with PBS, and a neural cell dispersion (manufactured by Wako corporation) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, cells were seeded at 8.0X10 ⁵ cells/well on a 12-well Transwell collagen gel. The initial 3 days were incubated in the presence of 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR 99021. After 3 days of inoculation, medium exchange was performed with serum-free medium (NucT medium) without Y-27632, SAG and CHIR99021 for 3-4 days 1.

For retinal cell sheets cultured for 37 days and 44 days of differentiation on day 63 and day 70 for re-aggregate formation, exfoliation was performed by treatment with collagenase (Roche company) at 37 ℃ for 30 minutes. The detached retinal sheet was cut into a long strip by forceps and scissors, and an implant for implantation having a length of about 1.8cm was prepared (fig. 47).

For the prepared grafts, glass Pasteur pipettes were used to graft under the retina of immunodeficient retinal deficient rats (SD Foxn).

The eyes of rats 1 year after transplantation were removed and fixed with 4% pfa. Observation with a fluorescent microscope and a fluorescence microscope (Keyence BZ-X810) confirmed Crx: : venus positive grafts survived (FIG. 48).

From this, it was found that the re-flaked retinal sheet survived after implantation.

Example 17 sorting Studies ]

Aggregates of the suspension culture prepared in example 1 were washed with PBS 14 to 25 days after the start of the suspension culture, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing, and after target cell populations were sorted by FSC and SSC using a cell sorter ARIAII (BD company), rx was collected by pipetting: : venus positive fraction (FIG. 49).

The cells thus obtained were inoculated into 12-well Transwell (Corning Co.) coated with laminin 511-E8 in 1.6 to 8.0X10- ⁵ cells per 1 well. As a control, unsorted pieces were also produced. The initial 3 days were incubated in the presence of 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR 99021. After 3 days of inoculation, medium exchange was performed with serum-free medium (NucT medium) without Y-27632, SAG and CHIR99021 for 3-4 days 1. Observations were made with a fluorescent light microscope at day 26 post inoculation (fig. 50).

In addition, the passage on day 82 of differentiation was sorted by Rx: : after the Venus purified cell pellet was fixed with 4% PFA, immunostaining was performed in pellet form without preparing a section. Immunostaining was performed with DAPI and anti-restorer antibodies (Proteintech). As a control, unsorted retinal sheets were also stained (fig. 51, 52, 53). Thereafter, frozen sections were prepared, and immunostaining was performed with DAPI and an Anti-Ki 67 Antibody (BD Co.), an Anti-Chx 10 Antibody (trade name: anti CHX10 Anti-body, santa Cruz Co.), and an Anti-CRX Antibody (TaKaRa Co.). (FIG. 53).

As a result, if not for Rx: : the Venus positive fraction was sorted and inoculated directly, and a block of RPE cells was observed in a portion. However, RPE cells may be removed for visual confirmation. On the other hand, for Rx: : when Venus positive fractions were sorted, it was observed that Rx could be made: : venus positive retinal sheets. It was further confirmed that most of the RPE cells could be removed (FIGS. 49 and 50). However, even if the sorting is performed, RPE cells may be mixed in, and it is not clear that the RPE cells cannot be completely removed by the sorting or that differentiation occurs after the sorting. However, it was confirmed that the mixed RPE cells were not mixed with the neural retinal tissue (fig. 52). Since it can be confirmed visually, RPE cells can be removed.

It was also confirmed that the sorted retinal sheets were able to differentiate restorer protein positive visual cells without being inferior to the control, non-sorted retinal sheets (fig. 51, 53). From the Z Stack analysis and immunohistological analysis of the sections, it was confirmed that the layer structure of retinal tissue was reformed and that Chx 10-positive and Ki 67-positive retinal precursor cells, crx-positive visual cell precursor cells were present (fig. 53).

It was thus observed that by sorting out the target cell population with FSC and SSC, with Rx: : venus, as an indicator, sorted to remove most of the RPE cells and differentiate retinal cells without problems upon re-flaking.

Example 18 exploration of factors that maintain retinal Properties upon re-flaking

Aggregates (NR) on day 25 after the start of suspension culture prepared in example 1 were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing, using a cell sorter ARIAII (BD company) at Rx: : venus is used as an index to implement sorting. For the sorted cells, Y-27632, CHIR99021, SAG were added to 100. Mu.L NucT and inoculated in a manner of 5X 10 ⁴ cells per 1 well in a 96-well glass bottom plate (the very company, calif.) coated with laminin 511-E8 using Easy iMatrix. Simultaneously with the inoculation, proteins shown in table 1 or low molecular compounds shown in table 2 were added.

TABLE 1

TABLE 2

After 3 days of inoculation, medium exchange was performed in order to maintain the concentrations of the low molecular compounds or proteins described in tables 1 and 2 as well. Thereafter, medium exchange was performed on day 7 with NucT's medium containing no low-molecular compounds or proteins as described in tables 1 and 2. 10 days after inoculation, the mixture was fixed with 4% PFA and subjected to a fluorescence microscope (BZ-X810, manufactured by Keyence Co.) to obtain a sample at Rx: : venus is used as an index to evaluate the sorted cells. As a result, in the protein, the FGF2, FGF4, FGF 8-added tablets were observed as Rx compared to PBS as a control: : venus has high expression intensity (FIG. 54). In addition, of the low molecular compounds, the sheet to which IWR1 endo was added observed Rx: : the expression intensity of Venus was high (FIG. 55). Further, the concentration dependence was confirmed, and when FGF8 was added at 100. Mu.g/mL, rx was observed: : venus expressed the highest intensity (FIG. 56).

From this, it can be seen that Rx can be maintained by activating FGF signal just after seeding cells: : venus positive cell population.

Example 19 confirmation of aggregate reformation and retinal differentiation by FGF8 addition >

Aggregates (NR) produced in example 1 on day 24 after the start of suspension culture were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing, using a cell sorter ARIAII (BD company) at Rx: : venus is used as an index to implement sorting. For the sorted cells, 2.0 to 5.0X10 ⁴ cells per 1 well were seeded on low adhesion V plates. As controls, a group to which FGF8 was not added (FGF 8-), and a group to which no sorting was performed (no sorting) were also set.

The medium exchange was performed 1 time 3 to 4 days. FGF8 was also added during medium exchange. Observation with a microscope on day 10 after re-inoculation resulted in Rx observed in the group without sorting, FGF 8-: : venus negative cell mass or RPE cells (FIG. 57). On the other hand, these cell mass reductions were observed in the group to which FGF8 was added (FGF 8+). In the group with sorted, FGF8-, RPE cells were observed locally. Since RPE cells were removed by sorting, rx was suggested: : a fraction of the population of Venus positive cells differentiated into RPE cells. On the other hand, rx is formed in the sorted, FGF8+ group: : venus positive aggregates, no RPE cells, rx were observed: : venus negative blocks.

From these results, RPE cells or Rx can be reduced by adding FGF 8: : differentiation of Venus negative cells.

Further, the aggregates on day 54 of the culture for re-formation of the aggregates (day 30 after re-inoculation) were washed with PBS, dispersed into single cells using a nerve cell dispersion (manufactured by Wako corporation), and then formaldehyde-fixed with a fixing buffer (BD corporation). After that, after washing with Perm/washing buffer (BD Co.), staining was performed with anti-CHX 10-Alexa Fluo 647 conjugated antibody (Santacruz Co.), anti-Sox 2-BV421 antibody (Biolegend Co.), anti-Ki 67-Alexa Fluo 647 conjugated antibody (BD Co.), anti-CRX-Alexa Fluo 647 conjugated antibody (Santacruz Co.). After washing the antibody with Perm/wash buffer (BD Co.), the antibody was measured with FACSCantoII (BD Bioscience Co.) and analyzed with FlowJo. As a control, neural retinal tissues having the same differentiation days as that of the non-reorganized neural retinal tissues were also set.

As a result, in the group without sorting and without FGF8 added, more Rx was observed: : a Venus negative cell population having Rx: : venus positive rate was low. On the other hand, it was shown that in the group to which FGF8 was added, although not sorted, there was Rx: : the Venus positive rate tended to be high (FIG. 58).

In addition, the group to which FGF8 was not added at the time of sorting also had Rx: : the Venus positive rate tended to be high (FIG. 58). On the other hand, the group to which FGF8 was added after sorting had a tendency that the proportion of the neural retinal precursor cells that were Chx10 positive and Sox2 positive was high and the proportion of the visual cell precursor cells that were Crx positive was low (fig. 59).

From these results, it can be seen that by adding FGF8 as well as sorting, RPE, rx: : differentiation of Venus negative cells gives uniform and good aggregates.

< Confirmation of Effect of FGF8 upon Reflaking of example 20 >

Aggregates (NR) produced in example 1 on day 24 after the start of suspension culture were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing, using a cell sorter ARIAII (BD company) at Rx: : venus is used as an index to implement sorting. For the sorted cells, 2.0X10 ⁵ cells per 1 well were suspended in 24-well Transwell (Corning Co.) and inoculated in NucT medium containing Y-27632, CHIR99021 and SAG, FGF 8. As a control, a group to which FGF8 was not added (FGF 8-) was also set.

The medium exchange was performed 1 time 3 to 4 days. FGF8 was also added during medium exchange. Plates were observed under a fluorescent microscope after 63 days of differentiation and 40 days after re-inoculation (FIGS. 60 and 61). In addition, for FACS analysis, aggregates obtained after differentiation for 64 days and 41 days after re-inoculation were washed with PBS, dispersed into single cells using a nerve cell dispersion (manufactured by Wako corporation), and then fixed with formaldehyde using a fixing buffer (BD corporation). After that, the sample was fixed with a fixing buffer (BD Co.) and washed with a Perm/washing buffer (BD Co.), and then stained with an anti-CHX 10-Alexa Fluo 647 conjugated antibody (Santacruz Co.), an anti-Sox 2-BV421 conjugated antibody (Biolegend Co.), an anti-Ki 67 antibody (BD Co.), and an anti-CRX-Alexa Fluo 647 conjugated antibody (Santacruz Co.). After washing the antibody with Perm/wash buffer (BD Co.), the antibody was measured with FACSCantoII (BD Bioscience Co.) and analyzed using FlowJo. As a control, a neural retinal tissue was also set up (fig. 62) which had not been reorganized and had the same differentiation days.

As a result, in the case of the tablets to which FGF8 was not added after sorting, more Rx was observed: : a Venus negative cell population having Rx: : venus positive cell clusters tend to be present on the block in the peripheral portion of the well. On the other hand, in the case of tablets to which FGF8 was added after sorting, rx: : venus positive cells were uniformly present throughout the wells, and good plates were produced (FIGS. 60 and 61). In the case of the tablet to which FGF8 was not added, a black RPE was observed (fig. 61, arrow), whereas in the case of the tablet to which FGF8 was added, almost no black RPE was observed.

In addition, in FACS analysis, when no FGF8 tablet was added, rx: : the Venus positive rate was reduced to 60%, while the FGF 8-added group was 95% or more, and it was found that the positive rate equivalent to the neural retinal tissue could be maintained (fig. 62). Furthermore, the ratios of the neural retinal precursor cells positive for Chx10 and Sox2 and the visual cell precursor cells positive for Crx were comparable to those of the neural retinal tissue. Has Rx: : venus positive and Ki67 positive proliferative retinal lineage cells tended to be more abundant than neural retinal tissue (FIG. 62).

From these results, it can be seen that RPE, rx can be reduced by adding FGF8 after sorting when re-flaking is performed: : the Venus negative cells differentiated to give uniform and good plates.

Example 21 confirmation of time-lapse change after inoculation and Reflaky preparation on Transwell >

Aggregates on day 27 after the start of suspension culture, which were prepared as in example 1, were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37 ℃, single cells were dispersed by blowing. After dispersion, in a 24-well Transwell coated with laminin 511-E8, 2.0X10 ⁵ cells per 1 well were suspended in 200. Mu.L serum-free medium containing 10. Mu. M Y-27632, 300nM SAG and 3. Mu.M CHIR99021, 100ng/mL FGF8, and incubated at 37℃for 3 days with 5% CO ₂. After 3 days of inoculation, medium exchange was performed with serum-free medium (NucT 0) free of Y-27632, SAG and CHIR99021, FGF8 for 3-4 days 1.

After 3 days, 6 days, 9 days, 12 days, 22 days, 40 days (dd 30, dd33, dd36, dd39, dd49, dd 67) of culture for re-forming aggregates, the resulting cell sheets were fixed with 4% pfa, and then were subjected to a fluorescent light microscope and a fluorescent microscope at Rx: : venus as an indicator observe the fragmentation and as a result, rx: : venus positive retinal cells proliferated and formed a sheet with thickness (FIG. 63). In particular, after 22 to 40 days, it was confirmed that the whole quilt Rx: : venus positive cells covered thick cell sheets.

From these results, it was observed that a reorganized retinal sheet was obtained after 22 to 40 days, which was useful for transplantation, analysis, and the like.

Example 22 RPC surface antigen screening-comparison of hES cells with NR >

Suspension culture for differentiation induction was started as described in example 1 using the above cells. SAG 300nM was added at the beginning of differentiation induction. Aggregates (NR) at day 19 and day 26 after the start of suspension culture were washed with PBS, and a neural cell dispersion (Wako Co.) was added. After incubation at 37 ℃, the cells were dispersed into single cells by blowing, and surface antigen screening (Alexa 647, a group of fluorochrome-conjugated antibodies) was performed using the surface antigen screening kit MACS (registered trademark) MARKER SCREEN, human (Miltenyi). As a control, human ES cells (hescs) were stained. The number of markers used in the screening was 371 in total (fig. 64 to 69).

In FACS analysis, MACS Quant10 (Miltenyi Co.) was used for measurement and FlowJo was used for analysis. Gating and analysis were performed using the groups indicated by FSC and SSC shown in fig. 65. First, correct staining and analysis was confirmed with E-cadherin, SSEA-4, SSEA-5 expressed in hESC and disappeared upon differentiation (FIG. 66). The analysis results are summarized in fig. 64. Note that, "hESC: high "refers to high or expressed levels of expression in human ES cells," hESC: low "means that the expression level is low or not in human ES cells," NR: high "means high expression level in neural retinal cells," NR: low "refers to low or no expression levels in neural retinal cells.

Non-expression (or expression) in hescs, 19 th or 26 th day after suspension culture start at Rx: : the results suggest that CD39, CD73, CXCR4, and thus CD29, CD49b, CD49c, CD49f, CD57, CD82, CD90, CD200 may be able to distinguish Rx: : markers for Venus positive cells with other cells (Rx:: venus negative cells, hESC) (FIG. 67).

Further, FACS analysis of CD39, CD73, CD184 (CXCR 4) expression of dd18, dd25, dd53, dd81 (d 18, d25, d53, d81 in FIG. 68) after the start of suspension culture was performed using a mouse IgG1-APC conjugated antibody (Miltenyi Co.), an anti-CD 39-APC conjugated antibody (Miltenyi Co.), an anti-CD 73-APC conjugated antibody (Miltenyi Co.), and an anti-CD 184 (CXCR 4) -APC conjugated antibody (Miltenyi Co.).

As a result, it was found that the ratio of CD39 and CD73 expressing cells was highest at dd25, and the ratio of CD184 expressing cells was high at dd18 and dd 25.

From these results, it was found that when the surface antigens CD39, CD73 and CD184 were used for purifying the neural retina precursor cells, the suspension culture was preferably carried out 18 days after the start of the suspension culture and around the 25 th day (fig. 68).

< Analysis of expression of CD39 and CD73, CXCR4 in example 23 >

SAG 300nM was added at the beginning of suspension culture (at the beginning of differentiation induction), and BMP4 was not added on day 3 after the beginning of suspension culture, thereby preparing brain organoids. As a control, a group to which BMP4 was added was set. Fluorescence microscopy was performed on brain organoids at day 26 after the start of the suspension culture thus prepared (fig. 70). As a result, differentiation of Rx was observed in the BMP 4-added group: : venus positive retinal tissue, on the other hand, differentiation of Rx was observed in the group without BMP 4: : venus negative neuroepithelial cells. Further, these aggregates were fixed with 4% PFA, and cut into pieces, stained with DAPI and an anti-FoxG A antibody (TaKaRa Co., ltd.) and observed with a fluorescence microscope (BZ-X810, keyence Co.). As a result, it was confirmed that the BMP 4-added group was FoxG negative, while the BMP 4-not-added group was positive for FoxG invasion, and it was confirmed that the brain organoids were differentiated (fig. 71).

Brain organoids on day 25 after the start of the suspension culture thus prepared were washed with PBS, and a nerve cell dispersion (manufactured by Wako Co.) was added thereto. After incubation at 37℃the cells were dispersed into single cells by blowing, stained with anti-CD 39-APC conjugated antibody (Miltenyi Co.), anti-CD 73-APC conjugated antibody (Miltenyi Co.), and anti-CXCR 4-APC conjugated antibody (Miltenyi Co.), the proportion of the expressed cell population was determined using FACSCantoII (BD Bioscience Co.) in the FACS assay, and analyzed using FlowJo.

As a result, it was observed that CD39 and CD73 were not expressed in brain organoids, while CXCR4 was expressed in most cells of brain organoids (fig. 72).

From this, it was confirmed that Rx can be isolated by differentiating from brain organoids using cell populations positive for CD39, positive for CD73 and negative for CXCR4 as indicators: : venus positive cell population.

Example 24 CD39 expression analysis

At the start of differentiation induction, the morphology of aggregates (NR) produced at the 26 th day after the start of suspension culture by adding SAG in (1) 0nM, (2) 30nM, and (3) 300nM was observed with a microscope. As a result, it was confirmed that aggregates were larger when SAG 0nM and SAG 30nM were added, and that the layer structure was also clearer than when SAG 300nM was added at the start of suspension culture (dd 0) (fig. 73).

Further, for the above aggregates, the neural cell dispersion was used to disperse into single cells, and FACS analysis of CD39 and CXCR4 expression was performed with an anti-CD 39-APC conjugated antibody (Miltenyi) and an anti-CXCR 4-APC conjugated antibody (Miltenyi). The proportion of the expressed cell population was determined using FACSCantoII (BD Bioscience) and analyzed using FlowJo. These aggregates were fixed with 4% pfa, and after slicing, stained with an anti-ALDH 1A1 antibody (R & DSystems), an anti-CoupTF A1 antibody (PPMX), an anti-LHX 2 antibody (Millipore), an anti-Chx 10 antibody (ExAlpha), an anti-Pax 2 antibody (covancer), and an anti-NKX 2.1 antibody (Leica).

As a result, it was observed that CD39 was expressed and CXCR4 was expressed low in aggregates to which SAG 300nM was added at the beginning of suspension culture (dd 0) (fig. 74). In addition, by adding SAG to dd0, a decrease in the expression of dorsal marker ALDH1A1 and an increase in the expression of ventral marker CoupTF were observed (fig. 75, 76). It was also found that the expression level of Rx in the cell aggregates was uniform when SAG stimulation was not applied (FIG. 77). However, in the case of LHX2, chx10 and Pax2, the addition of 300nM aggregates also confirmed expression in the region where Rx expression was low, and NKX2.1 was negative, and thus neural retinal tissue was confirmed (fig. 78 and 79).

From this, it was found that by adding SAG 300nM at the beginning of differentiation induction, a population of CD 39-positive and CXCR 4-negative neural retinal precursor cells could be obtained.

Example 25 CD39 expression enhancement study

To aggregates (NR) prepared by adding SAG 300nM at the beginning of differentiation induction (dd 0), 10 neural retinal tissues were transferred to a low-adhesion suspension culture dish 60mmDish (manufactured by Sumitomo electric Co., ltd.) on the 17 th day after the beginning of suspension culture, 1mM, 0.2mM adenosine (A2A receptor agonist, manufactured by Sigma Co.), 1mM, 0.2mM, 0.04mM AMP-PNP (manufactured by Tocris Co., ltd.), 5mM, 1mM, 0.2mM, 0.04mM ATP disodium salt (manufactured by Tocris Co., ltd.), 1. Mu.M, 0.2. Mu.M, 0.04. Mu.M CGS21680 (adenosine A2A receptor agonist, manufactured by Tocris Co., ltd.) were added to 4mL NucT0 medium, and the changes in expression of CD39 and CXCR4 were measured by using mouse IgG1-APC conjugated antibodies (Miltenyi Co., miltenyi), anti-CD 39-BV conjugated antibodies (BD Bioscience Co., ltd.) and anti-CXCR 4-conjugated antibodies (manufactured by Flotei, manufactured by Flotex Co., ltd.) on the 25 th day after the beginning of suspension culture. As a control, a DMSO addition group was set.

As a result, ATP and A2A receptor agonists were found to have the effect of enhancing CD39 expression and reducing CXCR4 expression (fig. 80 and 81).

From this, it is clear that the expression of CD39 can be enhanced by adding ATP and an A2A receptor agonist.

Example 26 transplantation of Islet-1 KO hESC-retinal sheets by CD39 sorting and FGF8 addition

For a composition having Rx: : human ES cells (KhES-1 strain, (non-patent document 3)) which had been genetically modified and from which the Islet-1 gene had been knocked out by the Venus reporter gene were cultured under feeder cells-free conditions according to the methods described in "SCIENTIFIC REPORTS4, 3594 (2014)" and WO 2018/097253. As feeder cells-free medium, stemFit medium (trade name: AK03N, manufactured by Weisu Co., ltd.) was used, and as a scaffold for replacing feeder cells, laminin 511-E8 (trade name, manufactured by Yu Co., ltd.) was used.

For aggregates obtained by differentiating with SAG 300nM at the beginning of suspension culture (at the beginning of differentiation induction, dd 0), aggregates (NR) at the 25 th day of suspension culture initiation were dispersed into single cells, stained with an anti-CD 39-APC conjugated antibody (Miltenyi Co.) at 37℃for 15 minutes, and then subjected to sorting using a cell sorter ARIAII (BD Co.) using CD39-APC as an index. The sorted cells were seeded at 8.0X10 ⁵ cells per 1 well in a 12 well Transwell coated with collagen gel. Y-27632 (Wako) and SAG (Enzo Co.), CHIR99021 (Wako Co.), 50ng/mL FGF8 (Wako Co.) were added at the time of inoculation. In addition, as a control, a group (FGF 8-) to which FGF8 was not added was also set.

The medium exchange was performed once for 3 to 4 days. FGF8 was also added during medium exchange. Observation under a microscope on day 73 after the initiation of suspension culture revealed that Crx was sparsely observed in the group to which FGF8 was not added: : in contrast to the Venus cell population, crx was observed in the group supplemented with FGF 8: : the Venus cell population was uniformly present (fig. 82).

For the thus-produced sheet, after the sheet was recovered by collagenase at day 88 after the start of suspension culture, the graft for transplantation was cut into long strips by using micro-scissors (fig. 82), and was transplanted under the retina of an immunodeficient retinal deficient rat (SD Foxn).

From this, it was confirmed that retinal sheets produced from cells sorted with CD39 can be transplanted.

Example 27 RPC surface antigen screening-comparison of brain organoids with NR >

Aggregates (NR) on days 24 to 26 after the initiation of suspension culture, which were prepared by adding SAG 30nM to the suspension culture at the initiation of suspension culture (at the initiation of differentiation, dd 0), were dispersed into single cells using a neural cell dispersion (Wako Co.), stained using a surface antigen screening kit MACS (registered trademark) MARKER SCREEN, and human (Miltenyi Co.) to perform surface antigen screening (Alexa 647, fluorochrome-conjugated antibody group). As a control, brain organoids prepared without BMP4 addition on day 3 after initiation of differentiation induction were stained.

FACS analysis was performed using MACS Quant10 (Miltenyi company) and analysis was performed using FlowJo. Exploring Rx in brain organoids that were not expressed (or expressed) and that were 24-26 days after suspension culture initiation: : the results for surface antigens expressed (or not) by the Venus positive population showed that CD9, CD15, CD49c, CD66b, CD69, CD82, CD164, epCAM, erbB2 (CD 340) may be distinguishable from brain organoids (fig. 83).

Additionally shown is, as a distinction Rx: : markers for Venus positive and negative cells, CD9, CD24, CD49c, CD90, CXCR4, epCAM may be useful (fig. 84).

< Confirmation of expression of CD9 over time >

Aggregates (NR) of dd4, dd11, dd18, dd25, dd32, and dd46 prepared by adding SAG 30nM to hESC and suspension culture at the beginning (at the beginning of differentiation induction, dd 0) were dispersed into single cells with a nerve cell dispersion (Wako Co.), stained with an anti-CD 9 antibody conjugated with APC (BioLegend Co.), and FACS analysis was performed after washing the antibody. MACS Quant10 (Miltenyi Co.) was used for the determination in FACS analysis and FlowJo was used for the analysis.

As a result, it was found that CD9 was expressed in hescs, but once the expression was lost on day 4 of initiation of differentiation, the expression was initiated around day 11 (d 11 in fig. 85), and the positive cell rates were increased around dd18 (d 18 in fig. 85) to dd25 (d 25 in fig. 85) and dd32 (d 32 in fig. 85). It was confirmed that, as the period of purification, dd11 and thereafter are preferable, and purification after dd18 to dd32 is more preferable (FIG. 85).

EXAMPLE 29 investigation of removal of target extracellular cells Using SSEA1 as an index >

Aggregates (NR) on day 25 prepared by adding SAG 30nM to the suspension culture at the beginning (at the beginning of differentiation induction, dd 0) were dispersed into single cells with a neural cell dispersion (Wako Co.) and stained with an anti-CD 9 antibody (BioLegend Co.) conjugated with APC, stained with an anti-SSEA 1 antibody (BioLegend Co.) conjugated with BV421, measured using FACSCantoII (BD Bioscience Co.) and analyzed using FlowJo.

As a result, it was found that the Rx positive cells in the aggregate as a whole were about 87%, but the Rx positive cells were increased to 94% when the CD9 positive cells were gated. It was also known that Rx positive cells increased to about 96% when gating for CD9 positive and SSEA1 negative cells (fig. 86).

From this, it was found that by combining SSEA1 with CD9, retinal lineage cells can be further purified.

< Study of combinations of example 30 CD9, CD90, CXCR4, SSEA1 >

Aggregates (NR) on day 25 prepared by adding SAG 30nM to 200 cells at the beginning of suspension culture (at the beginning of differentiation induction, dd 0) were dispersed into single cells with a neural cell dispersion (Wako Co.) and the cells were divided into 7 aliquots and stained for surface antigens at 4℃for 1 hour according to the following 7 conditions. (1) staining (purification by Rx:: venus) with anti-CD 9-APC conjugated antibody (BioLegend Co.), staining (3) with anti-CD 90-APC conjugated antibody (BD Co.), staining (4) with anti-CD 184 (CXCR 4) -APC conjugated antibody (Miltenyi Co.), staining (5) with anti-CD 9-APC conjugated antibody (BioLegend Co.) and anti-SSEA 1-BV421 conjugated antibody (BioLegend Co.), staining (6) with anti-CD 9-APC conjugated antibody (BioLegend Co.) and anti-CD 90-BV421 conjugated antibody (BioLegend Co.), and (7) staining with anti-CD 90-APC conjugated antibody (BD Co.) and anti-SSEA 1-BV421 conjugated antibody (BioLegend Co.). After staining, sorting was performed using the box shown in fig. 87 as an index by ARIA II (BD company). Cells recovered by sorting were subjected to FACS analysis with MACS Quant10 (Miltenyi corporation) to confirm that purification was possible using the target cell fraction (fig. 87). Determining Rx in the purified cell population: : ratio of Venus positive cells, results compared to before sorting, by (1) using Rx: : the purity of the samples sorted by the Venus positive fraction was highest, but the samples purified using CD9 positive and SSEA-1 negative fractions were not inferior to those purified using Rx: : samples purified by Venus, rx: : the Venus positive rate was high (FIG. 88, FIG. 89).

These sorted cells were seeded at 2.0X10 ⁵ cells per 1 well in a 24 well Transwell coated with laminin 511-E8. 10 mu M Y-27632 (Wako Co.), 300nM SAG (Enzo Co.), 3. Mu.M CHIR99021 (Wako Co.), and 100ng/mL FGF8 (Wako Co.) were added at the time of inoculation. The medium exchange was performed once 3 to 4 days after inoculation.

Bright field and fluorescent observations were made with a fluorescent microscope on days 1 and 12 after initiation of the re-flaking culture, and as a result, it was observed that any purified cells survived on the Transwell net on day 1 after inoculation (fig. 90). Further, in observation after 12 days, rx was used: : in contrast to some black RPE observed in samples from Venus positive purification, no black RPE was observed in CD9 purified discs of CD9 positive and CD9 positive/SSEA 1 negative (CD 9/SSEA 1) and CD9 positive/CD 90 positive (CD 9/CD 90) populations (fig. 91).

From this, it was confirmed that retinal sheets sorted using CD9 and SSEA1 negative or CD90 positive fractions were compared to those using Rx: : the separation of Venus positive was better able to remove the RPE fraction.

< Confirmation of time-dependent changes in expression of CD9 and SSEA1 >

Aggregates (NR) of dd4, dd11, dd18, dd25, dd32, dd46 prepared by adding SAG 30nM to hESC and suspension culture at the beginning (at the beginning of differentiation induction, dd 0) were dispersed into single cells with a nerve cell dispersion (Wako Co.), stained with an anti-CD 9-APC conjugated antibody (BioLegend Co.) and an anti-SSEA 1-BV421 conjugated antibody (BioLegend Co.), and FACS analysis was performed. The measurement was performed using FACSCantoII (BD Bioscience company) and the analysis was performed using FlowJo.

As a result, although hescs were positive for CD9 and negative for SSEA1, they became negative for CD9 and positive for SSEA1 on day 4 after the initiation of differentiation (fig. 92). Furthermore, it was found that the population positive for CD9 and negative for SSEA1 began to appear from day 11 of differentiation, and the proportion of the target population increased on days 18 and 25. Sustained expression was also measured on days 32, 46.

From this, it was confirmed that, as the period of purification, it is preferable to perform purification after dd11 (d 11 in fig. 92), particularly after dd18 (d 18 in fig. 92) to dd32 (d 32 in fig. 92) (fig. 92).

The 12-well Transwell retinal sheets prepared by sorting using CD9 and SSEA1 as described above and adding 10. Mu. M Y-27632, 3. Mu.M CHIR99021, 300nM SAG and 100ng/mL FGF8 were collected by a scalpel and scissors on the 48 th and 62 th days after the initiation of suspension culture, and then the transplant graft was cut into strips by a micro scissors and transplanted under the retina of an immunodeficient retinal deficient rat (SD Foxn) (not shown).

From this, it was confirmed that retinal sheets produced using cells sorted by CD9 and SSEA1 can be transplanted.

Example 32 study of RPE-retinal tissue Complex Using gelatin >

Aggregates (NR) produced in example 1 at 18 to 30 days after the start of suspension culture were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37℃single cells were dispersed by blowing and seeded at 4.0-8.0X10- ⁵ cells per 1 well in a 12 well Transwell. Y-27632 and SAG, CHIR99021 were added to NucT medium at the time of inoculation. Then, the medium was exchanged with NucT.sup.0 medium containing no Y-27632, SAG and CHIR99021 for 3 to 4 days at a time, and cultured for 1 month or more.

Aggregates obtained by differentiating RPE 80 days after the initiation of suspension culture prepared in example 1 were inoculated into a culture medium prepared by adding B27 (Gibco Co.), L-glutamine, 10ng/mL FGF2, SB431542 to a DMEM (SIGMA Co.) and F12 Ham (SIGMA Co.) medium in a culture dish coated with laminin 511-E8, and cultured. RPE proliferated and spread gradually after adhesion, so that only RPE was peeled off with a 1mL gun head and inoculated on other laminin 511-E8 coated dishes, thereby performing purification. After the expansion culture, RPE of 1.0X10 ⁶ cells was inoculated into 12-well Transwell coated with laminin 511-E8, and medium exchange was performed 1 time for 3 to 4 days, and culture was performed for 1 month or more.

The RPE sheet obtained by the above culture was subjected to an adhesion operation with BeMatrix LS-W gelatin (neo-field gelatin company) to a sheet-like retinal tissue. As a specific procedure, each piece was collected from the Transwell using forceps and scissors, and the sheet-like retinal tissue and the RPE pieces were fused with 10% (w/v) gelatin, then fused with 20% gelatin, and finally 30% gelatin was added to adhere the sheet-like retinal tissue to the RPE pieces. After adhesion, the mixture was quenched to 4℃and incubated for 20 minutes to be firmly recovered, whereby a composite sheet was produced in which a sheet-like retinal tissue and an RPE sheet were adhered (FIGS. 93 to 98).

The retinal tissue-RPE composite sheet thus fabricated was sheared with forceps and scissors. The composite sheet is first split in half and then slit into strips along the slit face (fig. 99). As a result, the two sheets can be cut out in a state of being adhered via gelatin. The cut composite sheet was observed for its cross section, and RPE with melanin was confirmed on one side and Rx on the opposite side: : venus positive sheet retinal tissue was filled with gelatin therebetween (FIG. 100). The cut composite sheet is not peeled off by suction and discharge operations, which are operations of transplanting, or suction and discharge operations are performed by using a 1mL gun head with a front end cut off. As a result, the composite sheet is not separated, and suction and discharge can be repeated (fig. 101).

From this, it was found that an implantable retinal tissue-RPE composite sheet can be produced by using a sheet-like retinal tissue, an RPE sheet and gelatin.

Example 33 study of RPE-retinal tissue Complex Using fibrin

Aggregates (NR) produced in example 1 at 18 to 30 days after the start of suspension culture were washed with PBS, and a neural cell dispersion (manufactured by Wako Co.) was added. After incubation at 37℃single cells were dispersed by blowing and seeded at 4.0-8.0X10- ⁵ cells per 1 well in a 12 well Transwell. Y-27632 and SAG, CHIR99021 were added to NucT medium at the time of inoculation. Then, medium exchange was performed with NucT.sup.0 medium containing no Y-27632, SAG and CHIR99021 for 3 to 4 days at a time, and the culture was performed for 1 month or more (FIG. 102).

Aggregates obtained by differentiating RPE 80 days after the initiation of suspension culture prepared in example 1 were inoculated into a culture medium prepared by adding B27 (Gibco), L-glutamine, 10ng/mL FGF2, SB431542 to DMEM (SIGMA) and F12 Ham (SIGMA) media in a iMatrix-coated culture dish, and cultured. RPE proliferated and spread gradually after adhesion, so the RPE was peeled off only with a gun head and inoculated on other iMatrix511 coated dishes for purification. After the expansion culture, RPE of 1.0x10 ⁶ cells was inoculated into a 12-well Transwell coated with iMatrix511, and medium exchange was performed once for 3 to 4 days, and the culture was performed for 1 month or more (fig. 102).

The adhesion operation of the patch-like retinal tissue obtained by the above culture to the RPE patch was performed using BOLHEAL tissue adhesion (curing company). As a specific procedure, each piece was collected from the Transwell using forceps and scissors, 100. Mu.L of fibrinogen was added to the sheet-like retinal tissue, 100. Mu.L of thrombin was added to the RPE sheet, and the pieces were removed after fusion (FIGS. 103 to 104). Again, 100 μl of fibrinogen was added to the sheet retinal tissue and 100 μl of thrombin was added to the RPE sheet (fig. 104). On the culture dish, the mesh side of the RPE sheet was placed on the culture dish side with the tip side facing upward, and the sheet-like retinal tissue was placed from above with the tip side facing toward the RPE side (fig. 105). After the adhesion, the resulting sheet was incubated at room temperature for 5 minutes to be firmly recovered, thereby producing an adhered sheet (fig. 106 to 107). The thus-produced retinal tissue-RPE composite sheet was turned over with forceps, and was set up and observed with the sheet-like retinal tissue facing downward and the retinal pigment epithelium sheet facing upward (fig. 108). The adhered Transwell mesh was peeled off (fig. 109).

From this, it was found that a retinal tissue-RPE composite sheet can be produced by using a sheet-like retinal tissue, an RPE sheet and fibrin.

Example 34 study Using CELLSHIFTER to remove unwanted gelatin between two sheets >

It is known that the composite sheet studied in example 32 contains a large amount of gelatin between two sheets and is thicker. Therefore, in order to extrude and remove unnecessary gelatin between two sheets in the compounding step, a compounding apparatus in which a passage through which gelatin escapes is formed has been studied.

2, 4, 5 Channels were formed in a bank shape using a silicon wafer on a slide glass, and 2 pieces CELLSHIFTER (Cell Seed company) with gelatin added in the middle were extruded from above to see whether or not gelatin flowed out.

As a result, it was found that in a device in which 5 channels were provided and silicon wafers were provided in a bank shape, the excessive gelatin flowed out most easily (fig. 110).

Example 35 study of producing a planarization sheet on a temperature-responsive Petri dish and peeling and transplanting ]

The aggregate (NR) produced in example 1 at days 18 to 30 was washed with PBS, and a nerve cell dispersion (Wako Co.) was added thereto. After incubation at 37℃the cells were spread into single cells by blowing and seeded at 8.0X10 ⁵ cells/well in 7 grades of Petri dishes (Cell seed Co.) coated with laminin 511-E8 at 24 wells. The medium exchange was performed once for 3 to 4 days, and after 30 days from the inoculation, a plate was formed thickly, and then cooled to room temperature and incubated for 2 hours for observation. As a result, in the temperature responsive dishes of 7-grade peeling degree, it was observed that the retinal sheets could be peeled off by setting to room temperature in the wells 2b, 2c, 3, and 4 and the plurality of wells (fig. 111).

From this, it was found that the retinal sheet could be recovered by culturing in a temperature responsive culture dish.

For this at Rx: : venus as an index, and a temperature responsive dish prepared by adding 10. Mu. M Y-27632, 3. Mu.M CHIR99021, 300nM SAG and 100ng/mL FGF8 was used as an index, and after incubating at 4℃for 1 hour and recovering at 62 days after the start of suspension culture, the graft for transplantation was cut into long strips using micro shears and transplanted under the retina of an immunodeficient retinal deficient rat (SD Foxn) (not shown).

From this, it was confirmed that the retinal sheet cultured on the temperature responsive culture dish could be transplanted.

Claims

1. A method for producing retinal tissue having an epithelial structure, which comprises the step of culturing a dispersed population of retinal cells in suspension or in adhesion in a medium containing a Wnt signal transduction pathway-acting substance,

The retinal line cell population contains 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells.

2. The method according to claim 1, wherein the Wnt signaling pathway-acting substance is 1 or more selected from the group consisting of CHIR99021, BIO, wnt2b and Wnt3 a.

3. The production method according to claim 1 or 2, wherein the medium further contains 1 or more substances selected from the group consisting of a ROCK inhibitor, an SHH signal transduction pathway-acting substance, and an FGF signal transduction pathway-acting substance.

4. The production method according to claim 3, wherein the ROCK inhibitor is 1 or more selected from the group consisting of Y-27632, fasudil (HA 1077), and H-1152.

5. The method according to claim 3 or 4, wherein the SHH signal transduction pathway-acting substance is 1 or more selected from the group consisting of SAG, PMA and SHH.

6. The production method according to any one of claims 3 to 5, wherein the FGF signal transduction pathway-acting substance is 1 or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF 8.

7. The method according to any one of claims 1 to 6, wherein the method comprises a step of performing an adhesion culture in the medium containing the Wnt signaling pathway-acting substance, and the retinal tissue having an epithelial structure is a sheet-like retinal tissue.

8. The method according to claim 7, wherein the adhesion culture is performed using a culture vessel coated with an extracellular matrix and/or a temperature-responsive polymer.

9. The method according to claim 8, wherein a culture surface of the culture vessel is coated with the temperature-responsive polymer, and an upper surface of the temperature-responsive polymer is coated with the extracellular matrix.

10. The production method according to claim 8 or 9, wherein the extracellular matrix is 1 or more selected from the group consisting of collagen, laminin, fibronectin, matrigel, and vitronectin.

11. The manufacturing method according to any one of claims 8 to 10, further comprising the step of: exposing the culture vessel coated with the temperature-responsive polymer to a temperature at which a property of the temperature-responsive polymer changes, thereby peeling the sheet-like retinal tissue from the culture vessel.

12. The manufacturing method according to any one of claims 1 to 11, comprising the steps of: dispersing a cell aggregate containing 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells to obtain the dispersed retinal line cell population.

13. The manufacturing method according to claim 12, wherein comprising the steps of: differentiation of pluripotent stem cells is performed to obtain the cell aggregate containing 1 or more cells selected from the group consisting of retinal precursor cells and optic cell precursor cells.

14. The manufacturing method according to any one of claims 1 to 13, further comprising the step of: increasing the proportion of retinal precursor cells contained in the dispersed retinal cell population prior to suspension or adhesion culturing the dispersed retinal cell population.

15. The method of claim 14, wherein the mixing or differentiation of retinal pigment epithelial cells is inhibited.

16. The manufacturing method according to claim 14 or 15, wherein the step of increasing the proportion of the retinal precursor cells includes the steps of: contacting the dispersed population of retinal lineage cells with a substance that binds to more than 1 antigen selected from the group consisting of CD9, CD39, CD90, and CXCR4, resulting in a population of cells expressing the antigen.

17. The method of manufacturing according to claim 16, wherein the step of increasing the proportion of the retinal precursor cells comprises the steps of: the dispersed retinal cell population is further contacted with a substance that binds to 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84, to obtain a cell population having an expression level of the antigen of the reference value or less.

18. The manufacturing method according to any one of claims 14 to 17, wherein the step of increasing the proportion of the retinal precursor cells includes the steps of:

19. The production method according to any one of claims 1 to 18, wherein the retinal precursor cells and/or the visual cell precursor cells account for 50% or more of the total number of cells contained in the retinal cell line population.

20. The method according to any one of claims 1 to 18, wherein the retinal precursor cells and/or the visual cell precursor cells account for 80% or more of the total number of cells contained in the retinal cell line population.

21. The production method according to any one of claims 1 to 20, wherein the dispersed retinal cell population is cultured in the medium containing the Wnt signaling pathway-acting substance from the start of the suspension culture or the adhesion culture.

22. The production method according to any one of claims 1 to 21, wherein the orientation of the cells in the epithelial structure is a direction substantially perpendicular to the layer direction.

23. The manufacturing method according to any one of claims 1 to 22, further comprising the step of: retinal tissue having an epithelial structure obtained by suspension culture or adhesion culture is cut into a size required for transplantation.

24. The production method according to any one of claims 1 to 23, wherein the epithelial structure is a multilayer structure.

25. A sheet-like retinal tissue comprising a retinal cell layer having a multilayer structure, wherein,

(2) The retinal cell-based layer having a multilayer structure contains 1 or more cells selected from the group consisting of retinal precursor cells, optic cell precursor cells and optic cells,

(3) In each of the layers of the retinal cell line, the cells are oriented in a direction substantially perpendicular to the layer direction, an

(4) The diameter is more than 8 mm.

26. The sheet-like retinal tissue of claim 25, wherein the retinal cell-based layer having a multilayer structure further contains sheet-like retinal pigment epithelial cells joined to the retinal cell-based layer, the retinal cell-based layer and the respective surfaces of the sheet-like retinal pigment epithelial cells are substantially parallel in tangential direction, the top end face of the retinal cell-based layer and the top end face of the sheet-like retinal pigment epithelial cells are opposed to each other, and the retinal cell-based layer and the sheet-like retinal pigment epithelial cells are joined by an adhesion factor existing therebetween.

27. The sheet-like retinal tissue of claim 26 wherein the adhesion factor is an extracellular matrix or a hydrogel.

28. The sheet-like retinal tissue of claim 27, wherein the adhesion factor is 1 or more selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, collagen type IV, heparan sulfate proteoglycan, and entactin.

29. The sheet-like retinal tissue of claim 27 wherein the adhesion factor is gelatin or fibrin.

30. A pharmaceutical composition comprising the sheet-like retinal tissue of any one of claims 25-29.

31. A method of treating a disorder based on retinal lineage cells or retinal tissue disorders or damage to retinal tissue, comprising the step of transplanting the sheet-like retinal tissue of any one of claims 25-29 to a subject in need thereof.

32. A method of increasing the proportion of retinal precursor cells in a population of cells comprising the step of contacting the population of cells containing retinal precursor cells with a substance that binds to more than 1 antigen selected from the group consisting of CD9, CD24, CD29, CD39, CD47, CD49b, CD49c, CD49f, CD57, CD73, CD82, CD90, CD164, CD200, CD340 and CXCR 4.

33. The method of claim 32, comprising the step of contacting said population of cells comprising retinal precursor cells with a substance that binds to more than 1 antigen selected from the group consisting of CD9, CD39, CD90 and CXCR 4.

34. The method of claim 32 or 33, further comprising the step of contacting the population of cells comprising retinal precursor cells with a substance that binds to more than 1 antigen selected from the group consisting of SSEA1, CD66b, CD69, and CD 84.

35. A cell population wherein at least 1 factor selected from the group consisting of CD9, CD39, CD90 and CXCR4 is positive and contains more than 90% of Rx positive retinal precursor cells relative to the total number of cells of the cell population.

36. The population of claim 35, wherein the retinal precursor cells are negative for 1 or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD 84.